Regulation of neurotransmitter vesicles by the homeodomain protein UNC-4 and its transcriptional corepressor UNC-37/groucho in Caenorhabditis elegans cholinergic motor neurons

Abstract

Motor neuron function depends on neurotransmitter release from synaptic vesicles (SVs). Here we show that the UNC-4 homeoprotein and its transcriptional corepressor protein UNC-37 regulate SV protein levels in specific Caenorhabditis elegans motor neurons. UNC-4 is expressed in four classes (DA, VA, VC, and SAB) of cholinergic motor neurons. Antibody staining reveals that five different vesicular proteins (UNC-17, choline acetyltransferase, Synaptotagmin, Synaptobrevin, and RAB-3) are substantially reduced in unc-4 and unc-37 mutants in these cells; nonvesicular neuronal proteins (Syntaxin, UNC-18, and UNC-11) are not affected, however. Ultrastructural analysis of VA motor neurons in the mutant unc-4(e120) confirms that SV number in the presynaptic zone is reduced ( approximately 40%) whereas axonal diameter and synaptic morphology are not visibly altered. Because the UNC-4-UNC-37 complex has been shown to mediate transcriptional repression, we propose that these effects are performed via an intermediate gene. Our results are consistent with a model in which this unc-4 target gene ("gene-x") functions at a post-transcriptional level as a negative regulator of SV biogenesis or stability. Experiments with a temperature-sensitive unc-4 mutant show that the adult level of SV proteins strictly depends on unc-4 function during a critical period of motor neuron differentiation. unc-4 activity during this sensitive larval stage is also required for the creation of proper synaptic inputs to VA motor neurons. The temporal correlation of these events may mean that a common unc-4-dependent mechanism controls both the specificity of synaptic inputs as well as the strength of synaptic outputs for these motor neurons.

Fig. 1.

UNC-4:: GFP is expressed in four classes of ventral cord motor neurons (DA, VA, VC, and SAB). Theunc-4 promoter was fused to GFP for expression in transgenic animals (Pflugrad et al., 1997). A, Left lateral view showing a representative VA motor neuron with anteriorly directed axon in the VNC and a DA motor neuron with circumferential commissure and anteriorly directed axon in the dorsal nerve cord.B, Ventral view of L1 larva showing GFP accumulation in cell bodies of all nine DA motor neurons and in two SAB motor neurons (arrowheads). Axons are out of the plane of focus.C, Left lateral view of L2 larva showing GFP expression in all 12 VA motor neuron cell bodies. D, E, Ventral view of adult hermaphrodite showing GFP-positive VC4 and VC5 somata and VC1–VC6 axonal projections to vulval muscles (arrowhead). F, Left lateral view of GFP expression in anteriorly directed axonal projections (arrows) of SAB motor neurons. (Only two of four SAB processes are shown.) AVF processes in the nerve ring (arrowhead) and the I5 motor neuron (asterisk) in the posterior bulb of the pharynx also show UNC-4:: GFP expression. G, Three SAB motor neurons innervating ventral (SABVL, SABVR) and dorsal (SABD) muscles in the head. Images shown in D andF are flattened stacks of optical sections collected in the confocal microscope. Scale bars, 10 μm. Anterior is to theleft.

Fig. 2.

UNC-17 expression in motor neurons is decreased inunc-4 mutants. A, Left lateral view shows punctate pattern of UNC-17 antibody staining in the SAB-containing nerve cords in the head region of a wild-type animal.Arrows point to dorsal and ventral left sublateral processes. B, Punctate UNC-17 staining is reduced or eliminated in many of the head sublateral processes inunc-4(e120)-mutant animals. Note the absence of UNC-17 staining in the left ventral process containing SABVL.C, Ventral view of UNC-17 expression in wild-type VC axons that innervate the vulval muscles (arrowhead) is shown. D, UNC-17 staining of VC vulval projections is eliminated or reduced in unc-4(e120) mutants, but UNC-17 staining of other cholinergic processes in the VNC and ventral sublateral (SL) nerve cords is not noticeably affected. E–I, Left lateral views are shown. The unc-104 kinesin mutation was used to localize UNC-17 to the soma of ventral cord motor neurons (Hall and Hedgecock, 1991; Nonet et al., 1993). The nuclei of DA and VA motor neurons also express UNC-4:: GFP (see G) (Pflugrad et al., 1997). E, G, H, In animals that are wild-type for unc-4, strong UNC-17 antibody staining is visible in the cytoplasm of cholinergic motor neurons. F, I, In unc-4(e120) mutants, cytoplasmic UNC-17 staining is substantially reduced in theunc-4::gfp-expressing DA and VA motor neurons.H and I are enlargements of single VA neurons from E and F(arrows), respectively. Scale bars, 10 μm. Anterior is to the left.

Fig. 3.

Vesicular protein levels depend onunc-4 and unc-37 activity. Antibodies to RAB-3 (A), Synaptobrevin (B), and Synaptotagmin (C) were used to detect these vesicular proteins in the DA and VA motor neurons in wild-type and in unc-4- andunc-37-mutant animals. (Methods were as described for Fig. 2.) Vertical bars in the histograms indicate the percentage of DA and VA motor neurons showing intense antibody staining (white) plus the percentage of animals showing reduced or intermediate levels of staining (gray) (see Materials and Methods). At least 125 VA and DA neurons were scored for each strain and for each vesicular protein antibody. Allunc-4 and unc-37 mutants in these experiments are loss-of-function alleles.

Fig. 4.

UNC-18 expression is not altered inunc-4 and unc-37 mutants. A ventral view of the vulval area is shown. Anterior is to the left. Double antibody staining was performed with UNC-17 and UNC-18 antibodies. A, D, UNC-18 antibody (red) stains the axonal processes of the VCs in a diffuse pattern (arrow). B, E, UNC-17 (green) is detected in VC varicosities associated with vulval muscles (arrow). C, F,Merged images show overlapping UNC-17 and UNC-18 antibody staining (yellow). A–C, In wild type, the UNC-17 and UNC-18 antibodies colocalize to VC axonal projections. D–F, In unc-4 mutants, UNC-18 (D, F) but not UNC-17 (E, F) (arrow) is detectable in VC axonal processes. All images are stacked confocal sections and were merged in Photoshop (C, F). Scale bar,A–C,D–F, 10 μm.

Fig. 5.

Reduction of presynaptic vesicles in VA motor neurons in unc-4(e120). Vesicles were counted in EM photos of VA and VB cross sections showing PSDs (arrows). A, B, Presynaptic regions of VA (A) and VB (B) motor neurons in wild type are shown. C, D, Fewer vesicles are seen in the presynaptic region of VAs (C) versus VBs (D) in unc-4(e120). The circumference of individual VA and VB motor neuron processes is outlined with a heavy black border. Handwritten numbers on these prints were used to trace individual neurons through these serial sections for reconstruction of the wild-type andunc-4(e120) ventral nerve cords (White et al., 1986,1992). E, Plot of vesicles per PSD in VA and VB motor neurons in wild-type versus two unc-4-mutant animals [unc-4(1), unc-4(2)] is shown. Application of the Mann–Whitney statistical test indicates that the decreased number of vesicles in VA motor neurons inunc-4(e120) is significantly different from that of the VBs (*p < 0.005; **p ≪ 0.001). Error bars indicate SD.

Fig. 6.

Thrashing rates of L1 animals. The total number of body thrashes was counted during a 2 min period. A thrash was scored as a movement of the animal's body in either the dorsal or ventral direction. Error bars are ±SD. Values that are statistically different from wild type using the Student's t test (p ≪ 0.001) are marked with anasterisk. Results of experiments performed withunc-37(wd17) strains are denoted withgray shading.

Fig. 7.

unc-4 mutations do not affectunc-17-cha-1 promoter:: gfpexpression. The 3.2 kb unc-17-cha-1promoter:: gfp reporter gene is expressed in virtually all cholinergic neurons. A, B, Lateral view of GFP-positive DA and DB motor neurons in both wild-type andunc-4(e120) L1 larvae. C, D, Lateral view of L2 stage larva showing equivalent levels of GFP expression in postembryonically derived VA and VB motor neurons in wild-type and unc-4(e120) animals. Thearrowhead denotes AS motor neurons that are seen in both wild-type and unc-4 animals. Asterisksmark gut autofluorescence. Scale bars, 10 μm.

Fig. 8.

GFP-tagged Synaptobrevin is downregulated inunc-4-mutant motor neurons. The unc-4promoter was used to drive expression of SNB-1:: GFP (M. L. Nonet, 1999). A–D, Punctate SNB-1:: GFP staining is seen in SAB (A) and VC processes (C) in wild-type animals but is either reduced (B) or eliminated (D) in these neurons in unc-37(e262) mutants (arrows). Images are compressed stacks of optical sections obtained in the confocal microscope. E, Histogram of SNB-1:: GFP expression in VC and SAB motor neurons shows decreased GFP expression in unc-4(wd1) andunc-37(e262) mutants. Two sets of VC vulval synapses (anterior vs posterior) were scored per animal. Staining of these VC varicosities was usually correlated with staining in the adjacent VC4 and VC5 somata (n = 60 animals). Four SAB processes were scored per animal (n = 80 animals). Scale bars, 10 μm.

Fig. 9.

Model. A, UNC-4 and UNC-37 function together to repress an intermediate gene (gene-x) that negatively regulates vesicle number. In unc-4 orunc-37 loss-of-function mutants, gene-xis derepressed, thereby leading to decreased levels of synaptic vesicles and SV proteins. B, In wild-type animals, VA motor neurons receive input from specific command interneurons (interneuron A). In unc-4 andunc-37 mutants, VA motor neurons exhibit a reduced number of SVs and receive input from a different set of presynaptic partners (interneuron B) (White et al., 1992) (Hall, German, and Miller, unpublished observations).