Figure 2.
unc-3 Controls SAB Motor Neuron Synapse Formation(A) Schematic of SAB and other classes of cholinergic MNs (DA, DB, VA, VB, AS, VC) in C. elegans. (B) Diagram of a cross-section of the head showing SAB axons (orange, blue, green), head muscle (light gray), and pharyngeal muscle (dark gray). (C) Left view of the head showing the synapses (black dots) of SABVL onto ventral muscle and the synapses (black dots) of left axon of SABD onto dorsal muscle. Anterior is left. (D)
unc-3 is expressed in SAB neurons during larval (L1 and L4) stages. The transgene otIs476[
glr-4prom::TagRFP] was used as a marker of SAB neurons.
unc-3 expression was monitored using a fosmid-based gfp reporter. Expression of
unc-3 was also detected in SABV and SABD neurons during adult stages (data notshown). Scale bar, 5 um. n > 10. (E) Synaptic defects at the SAB innervation zones of
unc-3(
e151) and
madd-4(
kr249) mutants. In wild-type (WT) SABD synapses, the presynaptic zone marker SYD-1 is juxtaposed to the AChR marker (UNC-29) expressed in head muscle (20/20 animals examined). In
unc-3(
e151) mutants, the SABD presynaptic zones are formed but are abnormally clustered as evident by SYD-1 fluorescence. Using SYD-1::GFP as a marker, we observe enlarged, abnormally clustered presynaptic zones in 11/20 (55%) SABD axons in
unc-3 mutants compared to 1/20 (5%) SABD axons in WT animals. In the SABV of
unc-3(
e151) mutants, we barely observe any SYD-1 puncta (20/20 animals). Detailed quantification of SYD-1 positive puncta is provided in (J). Post-synaptic clustering of AChR (UNC-29::RFP) is significantly reduced in the SABD and never observed in the SABV neurons of
unc-3(
e151) mutants (20/20 animals examined). The clustering of AChR in
madd-4(
kr249) mutants is dramatically reduced in SAB synapses, while the presynaptic zones (labeled by SYD-1::GFP) are normally generated (20/20 animals examined). The line of faint small AChR clusters in SABV of
unc-3 and
madd-4 mutants corresponds to the boundary of muscle cells (not the SABV innervation zone), which often contains a small amount of extra-synaptic AChRs. As expected, this boundary of muscle cells is also evident in the WT SABV image. For optimal contrast SYD-1::GFP is depicted in red and UNC-29::RFP is in green. Scale bar, 10 um.(F) The degree of L-AChR clustering at the SABV and SABD innervation zones was evaluated by measuring UNC-29::RFP fluorescent intensity. The percentage(%) of fluorescent intensity of UNC-29::RFP was calculated along 55 mm of the SAB innervation zone as previously described in [4]. Error bars show SEM. We ran a Kruskal-Wallis nonparametric test followed by a Dunn's post test. *p < 0.05, ***p < 0.001. SABV: WT (n = 16),
unc-3 (n = 32),
madd-4 (n = 21); SABD, WT (n = 9),
unc-3 (n = 22),
madd-4 (n = 15). (G-J) Visualization of presynaptic boutons using a translational yfp reporter for synaptobrevin-1 (
snb-1). (G) A representative image of the boutons along the SABDL and SABVL axons is shown (left view) in WT and
unc-3(
e151) mutants. Scale bar, 5 um. n = 30. Presynaptic boutons often cluster abnormally in the SABD of
unc-3 mutants (indicated by brackets), while their number appears normal when compared to WT SABD. Using SNB-1::YFP as a marker, we observe enlarged,abnormally clustered presynaptic zones in 15/24 (62%) SABD axons in
unc-3 mutants compared to 3/20 (15%) SABD axons in WT animals. In SABV of
unc-3(
e151) mutants, the number of boutons is significantly reduced. Animals at the fourth larval (L4) stage were analyzed. Visualization of SAB active zones using an antibody for RIM protein. In SABD of
unc-3(
e151) mutants, the distribution of active zones is abnormal, as they often miscluster (indicated by brackets). However, the total number of active zones is not significantly reduced in the SABD of
unc-3(
e151) mutants. There is a significant reduction of active zones in the SABV of
unc-3(
e151) mutants. (H) Detailed quantification of SNB-1 positive boutons. (I) Quantification of RIM staining. (J) Detailed quantification of SYD-1 positive puncta. Animals at the first day of adulthood were analyzed. Each dot represents the number of SNB-1, RIM, or SYD-1 positive puncta along one SABD or SABV axon.***p < 0.001. N.S, not statistically significant. n = number of axons analyzed. Mean values are represented with a horizontal red line.