Caenorhabditis elegans innexins regulate active zone differentiation

Abstract

In a genetic screen for active zone defective mutants in Caenorhabditis elegans, we isolated a loss-of-function allele of unc-7, a gene encoding an innexin/pannexin family gap junction protein. Innexin UNC-7 regulates the size and distribution of active zones at C. elegans neuromuscular junctions. Loss-of-function mutations in another innexin, UNC-9, cause similar active zone defects as unc-7 mutants. In addition to presumptive gap junction localizations, both UNC-7 and UNC-9 are also localized perisynaptically throughout development and required in presynaptic neurons to regulate active zone differentiation. Our mosaic analyses, electron microscopy, as well as expression studies suggest a novel and likely nonjunctional role of specific innexins in active zone differentiation in addition to gap junction formations.

Figure 1.

Mutations in unc-7 specifically affect active zone differentiation. A–E, GABAergic active zone marker hpIs3 (Punc-25-GFP::SYD-2) expression in wild-type (hpIs3), unc-7(hp121), unc-7(hp121) plus panneuronal unc-7, unc-7(hp121) plus GABAergic unc-7 and unc-7(hp121) plus Cholinergic unc-7 L2 stage larvae. F, unc-7(hp121) animals carrying a genomic unc-7 construct, an UNC-7S construct, or constructs that express unc-7 pan-neuronally (PF25B3.3) in muscles (Pmyo-3), in GABAergic (Punc-25), or in cholinergic (Pacr-2) neurons were quantified for the bright GFP::SYD-2 puncta in DD5 motoneurons. The number in each bar graph shows the number of animals analyzed. G, H, Cholinergic active zone marker hpIs54 (GFP::SYD-2) expression in wild-type (hpIs54) and unc-7(e5) L2 stage larvae. I, J, Another GABAergic active zone marker hpIs61 (Punc-25-GFP::UNC-10) expression in wild-type (hpIs61) and unc-7(e5) L3 stage larvae. Scale bar, 5 μm. K, The distribution curve of the hpIs61 punctum width in L3 stage larvae of wild-type (solid line) and unc-7(e5) (dotted line) animals. x-axis represents the punctum width in micrometers; y-axis represents the density or the distribution of puncta in arbitrary units. N, The total number of puncta used for analyses: 577 for wild type, 490 for unc-7.

Figure 2.

Ultrastructural analysis of the active zone morphology in unc-7 animals. A–C, Serial electron micrographs of one GABAergic NMJs in wild-type animals (A series) and two GABAergic NMJs in unc-7 mutants (B and C series). White arrowheads mark the position of the active zone(s) in each synapse. Active zones in unc-7 animals can be larger (B) or less prominent (C). Scale bar, 100 nm. D, The distribution curve of active zone size in wild-type and unc-7 animals. x-axis represents the volume in micrometers cubed; y-axis represents the density of active zones in arbitrary units. E, F, Cholinergic and GABAergic nerve processes of a wild-type and a unc-7 animal reconstructed from serial electron micrographs. The varicosities along the axons are synapses, and the active zone(s) in each synapse are also traced and shown as either black or red dots. Asterisks mark abnormal synapses in unc-7 neurons with irregular active zones.

Figure 3.

UNC-7 is required for both synaptic transmission and synapse formation. A, unc-7(hp121) animals carrying a genomic unc-7 construct and UNC-7S construct or constructs that express unc-7 panneuronally (PF25B3.3) in muscles (Pmyo-3) or in GABAergic neurons (Punc-25) were assayed for aldicarb resistance. Shown here is the result of one representative set of aldicarb test result (Materials and Method). B, unc-7 is required for proper GFP::SYD-2(hpIs3) active zone marker morphology during a developmental stage coinciding with synaptogenesis. The percentage of adult unc-7(hs9) temperature-sensitive mutants showing the GFP::SYD-2 morphology defects when shifted between permissive and nonpermissive temperatures during different developmental stages. 22-15, unc-7(hs9) cold-sensitive mutants were shifted from 22°C (the permissive temperature) to 15°C (the nonpermissive temperature) at developmental stages indicated along the x-axis. Vice versa for 15–22°.

Figure 4.

UNC-7 localizes to perisynaptic regions along the dorsal nerve cord and the expression of UNC-7 in neurons is required for active zone differentiation. Specific UNC-7 antibody staining in nervous system (A), dorsal nerve cord (B), ventral nerve cord (C), and muscle (D; M, muscle; dc, dorsal cord). UNC-7 staining shows small discrete puncta, as well as to larger puncta occurring periodically along nerve cords (arrowheads). E, Staining of UNC-7 shows discrete puncta along the dorsal cord in the muscle. F–J, Coimmunostaining of UNC-7 (red) with presynaptic proteins (green) UNC-10 (F–F″), SYD-2 (G–G″), synaptic GFP markers UNC-49::GFP (H–H″), SYD-2::GFP (I–I″), and SNB-1::GFP (J–J″). K–K″, Live imaging of GFP::UNC-7 (green) and SNB-1::mRFP (red) in GABAergic neurons. Scale bar, 5 μm. L, An illustration of UNC-7 protein localization in neurons and muscles.

Figure 5.

The presence of gap junctions at the dorsal and ventral nerve cords of wild-type and unc-7(hp121) animals by electron microscopy analyses. A–B′, The cross sections of the dorsal (A, A′) and ventral (B, B′) nerve cords of a wild-type L1 larva. Circles present the cross sections of the nerve processes. m, Cross sections of the postsynaptic muscle arms that wrap around the nerve process bundles. Red arrows, Gap junctions; blue asterisks, examples of the uncharacterized “wannabe” junctions. C, The cross sections of the ventral nerve cord of a wild-type adult with gap junctions shown in red arrows and wannabe junctions in blue asterisks. D, The cross sections of the ventral and dorsal nerve cords of an unc-7(hp121) adult. Scale bars, 100 nm.

Figure 6.

UNC-9 innexin functions similarly to UNC-7 during active zone differentiation. A–C, GABAergic active zone marker GFP::SYD-2 expression in wild-type (hpIs3), unc-9(e101), and unc-7(e5) animals. D–H, Active zone marker GFP::UNC-10 expression in wild-type (hpIs61), unc-9(fc16), unc-7(e5), unc-7(e5)unc-9(fc16), and unc-1(e719) animals. I, Quantification of bright hpIs3 puncta in DD5 motoneurons of L2 and L4 larvae. The number of animals analyzed is shown in each bar graph. J–J‴, Coimmunostaining of anti-UNC-9 and anti-GFP antibody in UNC-7::GFP expressing animals showed colocalization between UNC-9 and UNC-7::GFP. J‴ provides an enlarged view of J″. K, The distribution curve of the GFP::UNC-10(hpIs61) punctum width (x-axis in micrometers) in L3 larvae of wild-type (solid line), unc-9(fc16), and unc-7(e5)unc-9(fc16) (dotted line) animals. N (the total number of puncta) = 577 (wild type), 378 (unc-9), and 237 (unc-7unc-9). Scale bars: A (for A–C), D (for D–H), J″ (for J–J″), J‴, 5 μm.