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The Journal of Neuroscience, March 15, 2001, 21(6):2001-2014
Regulation of Neurotransmitter Vesicles by the Homeodomain
Protein UNC-4 and Its Transcriptional Corepressor UNC-37/Groucho in
Caenorhabditis elegans Cholinergic Motor Neurons
Kim M.
Lickteig1,
Janet
S.
Duerr2,
Dennis L.
Frisby2,
David H.
Hall3,
James B.
Rand2, and
David M.
Miller III1
1 Department of Cell Biology, Vanderbilt University
Medical Center, Nashville, Tennessee 37232, 2 Program in
Molecular and Cell Biology, Oklahoma Medical Research Foundation,
Oklahoma City, Oklahoma 73104, and 3 Center for C. elegans Anatomy, Albert Einstein College of Medicine, Bronx, New
York 10461
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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 (~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.
Key words:
synaptic vesicles; cholinergic differentiation; C.
elegans; synaptic specificity; neural development; unc-4
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INTRODUCTION |
The function of motor neuron
circuits depends on synaptic transmission between specific cells.
Studies of the nematode Caenorhabditis elegans have revealed
that a homeodomain protein encoded by the unc-4 gene
controls the pattern of synaptic inputs to one class of motor neurons
in the ventral nerve cord (Miller et al., 1992
; White et al., 1992).
The strong backward movement defect that unc-4 mutants
display is correlated with the miswiring of VA motor neurons with
synapses from command interneurons normally reserved for their lineal
sister cells, the VB motor neurons. Expression of the UNC-4
protein in the VA motor neurons rescues this phenotype and thereby
establishes that unc-4 functions in the postsynaptic cell
(i.e., VA motor neuron) to block input from inappropriate presynaptic
partners (i.e., VB-type command interneurons) (Miller and Niemeyer,
1995). UNC-4 activity requires physical interaction with UNC-37, a
ubiquitously expressed Groucho-like transcriptional corepressor protein
(Miller et al., 1993; Pflugrad et al., 1997). Thus, we have proposed
that the creation of appropriate synaptic inputs to VA motor neurons
depends on UNC-4-UNC-37-mediated repression of "VB-specific genes"
(Winnier et al., 1999).
LacZ and green fluorescent protein (GFP) reporter genes have also
detected unc-4 expression in additional classes of motor neurons in the ventral nerve cord and in flanking ganglia (Miller and
Niemeyer, 1995; Pflugrad et al., 1997). Because these motor neurons
(e.g., DAs) are not miswired with improper inputs in unc-4 mutants (White et al., 1992), the functional significance of
unc-4 expression in these cells has been unclear. Here we
show that unc-4 and unc-37 mutations result in
decreased levels of synaptic vesicle (SV) proteins in all
unc-4-expressing motor neurons and that this deficit impairs
the function of these cells.
SVs are clustered near the presynaptic density (PSD) where they are
poised for rapid membrane fusion and release of neurotransmitter (for
review, see Calakos and Scheller, 1996). Specialized vesicular proteins are incorporated into the SVs to modulate these events. Vesicle exocytosis and recycling also depend on interactions with cytoplasmic and axonal membrane proteins. Expression of these synaptic
proteins is developmentally regulated and subject to both
transcriptional and post-transcriptional mechanisms of control (Bergmann et al., 1991; Lou and Bixby, 1993; Melloni et al., 1994; Petersohn et al., 1995; Deans et al., 1997; Smith et al., 1997). Additional homeostatic mechanisms of regulation are suggested by the
observation that neurons in many systems contain different types and
numbers of synaptic vesicles (White et al., 1986; Jia et al., 1993;
Merchan-Perez and Liberman, 1996; Jin et al., 1999). These differences
may indicate that SV levels are actively regulated in each of these
cell types to satisfy specific physiological requirements.
The evolutionary conservation of the secretory apparatus has
facilitated the use of genetic approaches in simple model systems to
discover new components of the mammalian exocytic pathway and to define
their modes of action (Rothman, 1994; M. Nonet, 1999; Lloyd et al.,
2000). Because UNC-4 and its cofactor UNC-37 (Pflugrad et al., 1997)
have been shown to function as transcriptional repressors (Winnier et
al., 1999), the decreased levels of vesicular proteins that we observe
in unc-4 and unc-37 mutants must result from an indirect mechanism of action; UNC-4 and UNC-37 may repress a target gene ("gene-x") that in turn exerts a negative effect on
vesicular protein levels. Furthermore, our results indicate that this
presumptive downstream gene acts via a post-transcriptional mechanism;
derepression of gene-x in unc-4 or
unc-37 mutants must either constrain SV biogenesis or
enhance SV turnover. These outcomes are strictly dependent on
unc-4 activity during embryonic and early larval stages of
motor neuron differentiation; SV protein expression does not require
unc-4 function in the adult. This finding parallels a
previous observation that the specificity of synaptic inputs to one
class of cholinergic motor neurons (e.g., VAs) also depends on
unc-4 function during a concurrent period of larval growth (Miller et al., 1992). The temporal coincidence of these events offers
the intriguing possibility that both the specificity of synaptic inputs
as well as the strength of synaptic outputs for these motor neurons may
depend on a common unc-4-regulated mechanism.
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MATERIALS AND METHODS |
Nematode strains. C. elegans were grown as
described (Brenner, 1974). The N2 (Bristol) strain was used as the
wild-type strain. All genetic experiments were performed at 25°C with
the exception that the unc-4(e2322ts) strain (Miller et al.,
1992) was grown at the permissive temperature of 16°C when noted.
Chromosomal integration of the unc-4 promoter-gfp
line (wdIs5) was achieved by 
irradiation (4000 rads).
Animals with gfp reporter genes [wdIs5,
unc-17-cha-1p:: gfp, and
unc-4p:: synaptobrevin-1 (SNB-1):: gfp] were anesthetized with 1%
tricaine and tetramisole and assessed as described previously (Miller
et al., 1999). The following mutant strains were used: in linkage group
I (LG I), unc-37(e262), unc-37(wd17),
bli-4(e937), unc-11(n2954), and
unc-13(e51); in LG II, bli-2(e768),
unc-104(e1265), unc-4 alleles e120,
wd1, e26, e2321, and
e2322ts; and in LG IV, dpy-20(e1282),
unc-17(e113), and cha-1(md39ts).
Genetics. unc-4 unc-104 double mutants were
constructed by first marking unc-4 with bli-2.
unc-104 was crossed into the bli-2 unc-4 strains,
and nonblister Unc-4 animals were picked to select for a
crossover event between bli-2 and unc-104. The
presence of unc-4 in the unc-4 unc-104 double
mutant was verified by a complementation test using
unc-4(e2322ts) males (Miller et al., 1993).
unc-37(wd17)-containing strains were constructed by crossing
bli-4 unc-37(wd17) animals (Miller et al., 1993) with
unc-4 unc-104 strains. The presence of the wd17
mutation in this strain was confirmed by the appearance of non-Unc-4
progeny from matings with unc-4(e2322ts) males at the
restrictive temperature (25°C).
UNC-18 antibody. The DNA sequence corresponding to amino
acids 18-584 of UNC-18 was amplified from a cDNA library by PCR and cloned into pRSETB (Invitrogen, San Diego, CA). A fusion protein with a
6×-His terminal tag was expressed with the Xpress System (Invitrogen)
and purified over a ProBond resin column. Purified fusion protein was
used to immunize two goats and two rabbits. Anti-UNC-18 was affinity
purified with the fusion protein coupled with methanol to
nitrocellulose filters (Duerr et al., 1999). Antiserum from one goat
(G247) gave the most specific signal on fixed C. elegans and
was used for this study.
Synaptotagmin antibody. A bacterially expressed fusion
protein containing amino acids 144-441 of the synaptotagmin
(SNT-1) protein was produced and purified as described
previously (Nonet et al., 1993) and used to immunize rabbits. The
resulting sera were affinity purified using the bacterial fusion protein.
Immunofluorescence. To evaluate antibody staining of the DA
and VA motor neurons, animals were synchronized using a Chlorox and
NaOH solution, and the resultant embryos were grown to the late L2
stage (Sulston and Hodgkin, 1988). Antibody staining of the DAs and VAs
was performed within 1 d of fixation to preserve the GFP
fluorescence that tended to fade with time. Adult animals were
antibody-stained for evaluation of vesicular and neuronal protein
expression in the VC and SAB motor neurons. All antibodies were diluted
in antibody buffer B, pH 7.2 (1× PBS, 0.5% Triton X-100, 1 mM EDTA, pH 8.0, and 0.1% BSA) (Duerr et al., 1999). Stained animals were placed in a mounting media containing 200 mg/ml
n-propyl gallate, 30 mM Tris, pH 9, 70% glycerol, and 20 µg/ml 4',6-diamidino-2-phenylindole.
For UNC-17 and choline acetyltransferase (ChAT) antibody staining,
animals were placed on a slide, immobilized by pressing down on a
coverslip, and frozen on dry ice for 30 min. After removal of the
coverslip (freeze-crack), the animals were fixed in methanol for 2 min
and then acetone for 4 min at 4°C. Samples were blocked for 1 hr at
room temperature (RT) with 10% goat serum. Incubation with a 1:200
dilution of monoclonal UNC-17 antibody or 100 µl of ChAT antibody
sera (J. Duerr and J. Rand, unpublished observations) was
performed for 4 hr to overnight at RT. Goat anti-mouse indocarbocyanine (Cy3) secondary antibody (1:400) (Jackson ImmunoResearch, West Grove,
PA) was incubated for 4 hr at RT.
Double antibody staining with UNC-18 and UNC-17 antibodies was
performed in a two-step process. Freeze-cracked animals were fixed for
2 min in methanol followed by 4 min in acetone. Animals were blocked in
1% BSA and then incubated with a 1:40 dilution of goat anti-UNC-18 at
4°C overnight. Secondary antibody of rabbit anti-goat Cy3 (1:200) was
then incubated for 4 hr at RT. Next, 10% goat serum was added to block
residual rabbit anti-goat Cy3-binding sites. Animals were then
incubated with UNC-17 mouse monoclonal antibody (1:200) for 4 hr at RT
followed by incubation with secondary antibody of goat anti-mouse
Cy2 (1:600) for 4 hr at RT.
Antibody staining with UNC-11 (Nonet et al., 1999) was performed on
freeze-cracked animals fixed for 10 min in methanol at 
20°C. UNC-11
antibody was incubated at 1:200 dilution overnight at 4°C. Secondary
antibody of goat anti-rabbit Cy3 (1:200) was incubated at RT for 4 hr.
Synaptotagmin antibody staining was performed on freeze-cracked animals
prepared as described above except that animals were subsequently fixed
in 2% paraformaldehyde on ice for 4 hr. Synaptotagmin antibody was
incubated for 4 hr at RT at a 1:50 dilution. Incubation with goat
anti-rabbit Cy3 secondary antibody (1:200) (Jackson ImmunoResearch) was
performed for 4 hr at RT.
For the remainder of the antibodies used in these experiments (see
below), animals were fixed with 0.4 ml of Bouin's solution (75 ml of
saturated picric acid, 25 ml of formalin, and 5 ml of glacial acetic
acid), 0.4 ml of methanol, and 0.01 ml of 
-mercaptoethanol followed
by incubations in primary antibodies for 2 hr at RT and secondary
antibodies for 2 hr at RT. Primary antibody combinations included
anti-Synaptobrevin (1:50) (Nonet et al., 1998) and monoclonal GFP
antibody (1:2000) (Clontech, Cambridge, UK); anti-RAB-3 (1:100) (Nonet
et al., 1997) and polyclonal GFP antibody (1:1000) (Molecular Probes,
Eugene, OR); and anti-Syntaxin (1:1000) (Saifee et al., 1998) and
anti-RAB-3 (1:50) (Nonet et al., 1997). Secondary antibodies used were
goat anti-rabbit Cy3 (1:500), goat anti-mouse Cy2 (1:200), goat
anti-mouse Cy3 (1:500), and goat anti-rabbit Cy2 (1:200) (Jackson ImmunoResearch).
Microscopy. Immunofluorescent micrographs were obtained in a
Zeiss Axioplan microscope and in a Zeiss LSM 410 confocal microscope.
The fluorescent intensity of individual DA and VA motor neurons was
defined by comparison with flanking neurons in the ventral nerve cord
(VNC) (i.e., VB, DB, and AS neurons). Staining intensity was
scored in three categories: "wild type," in which DA and VA staining was comparable with that of adjacent cholinergic neurons; "reduced," in which DA and VA fluorescent intensity was visibly less bright than that of the reference neurons; "none," in which staining was not detectable or very faint.
Electron micrograph analysis. Existing sets of serial
electron micrographs (EMs) of N2 (White et al., 1986) and of two
unc-4(e120) animals (White et al., 1992) were examined to
determine the numbers of presynaptic neurotransmitter vesicles in VA
and VB motor neurons. Randomly selected prints were digitized on a flat
bed scanner and recorded on compact disks. The number of vesicles in
the VA and VB motor neurons was counted in each image (Adobe
Photoshop). Sections that contained a thickening that resembled a PSD
were then used to calculate the number of vesicles per PSD. For N2, four VA neurons (40 PSDs) and three VB neurons (43 PSDs) were analyzed.
Two unc-4 animals were analyzed with a total of four VA
neurons (54 PSDs) and four VB neurons (41 PSDs). The Mann-Whitney test
was performed to determine whether the average number of vesicles per
PSD was statistically different in the VAs versus the VBs in wild-type
and unc-4-mutant backgrounds.
Thrashing assay. Adult animals were subjected to Chlorox and
NaOH solution to isolate embryos (Sulston and Hodgkin, 1988). Embryos
were placed on clean plates and incubated for 2 hr at 25°C. Hatched
L1 animals were placed in M9 buffer in a 96-well microtiter plate. The
number of times an active animal thrashed in a given direction was
counted in a 2 min interval (Miller et al., 1996).
unc-4:: SNB-1:: gfp transgenic
strains. Transgenic lines were obtained by injecting
pSB126.65 (unc-4:: SNB-1:: gfp)
(M. L. Nonet, 1999) and pMH86 [the dpy-20(+)
cotransformation marker] (Clark et al., 1995) at 25 ng/µl (Mello and
Fire, 1995). Standard genetic crosses were used to place the reporter
gene into unc-4(wd1) and unc-37(e262) backgrounds.
Temperature-shift experiments. UNC-17 antibody staining was
evaluated as a function of unc-4 activity in DA and VA motor
neurons. The double mutant unc-4(e2322ts) unc-104(e1265) was
used for all temperature-shift experiments. For the analysis of
unc-4(e2322ts) animals (Miller et al., 1992) at the
permissive temperature, animals were raised at 16°C and then
synchronized by Chlorox treatment (Sulston and Hodgkin, 1988). These
animals were grown at the permissive temperature until the early adult
stage. For analysis of unc-4ts at the restrictive
temperature, animals were raised at 25°C, synchronized, and grown to
the early adult stage. For the temperature-shift experiments, animals
were raised at the first temperature ("embryo"), synchronized,
shifted to the second temperature, and grown until the end of the L2
stage (determined by the appearance of the postderid cells;
"L1-L2") (Sulston and Horvitz, 1977); these animals were then
grown at the third temperature until the early adult stage ("adult"). UNC-17 antibody staining for these animals was performed as described above.
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RESULTS |
UNC-4 is expressed in cholinergic motor neurons
The homeodomain protein UNC-4 is expressed in specific classes of
motor neurons. In the ventral nerve cord, UNC-4 is expressed in A-type
motor neurons (the VAs and DAs) (Miller and Niemeyer, 1995) and in the
VC motor neurons (Fig. 1). The VAs extend
anteriorly directed axons within the ventral nerve fascicle, whereas
the DAs send out commissural processes that also project anteriorly after entering the dorsal nerve cord (White et al., 1986). There are
six VC motor neurons that innervate the vulval muscles in the
midventral region (White et al., 1976, 1986). unc-4 is
initially expressed in the VCs at the onset of vulval morphogenesis in
the mid-L3 larval stage (data not shown). UNC-4 is also expressed in
three SAB motor neurons (SABVL, SABVR, and SABD) (Miller and Niemeyer, 1995) that send out four anteriorly directed processes in the
dorsal and ventral sublateral nerve cords adjacent to muscles in the
head (White et al., 1986).
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Figure 1.
UNC-4:: GFP is expressed in four classes
of ventral cord motor neurons (DA, VA, VC, and SAB). The
unc-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 and
F are flattened stacks of optical sections collected in
the confocal microscope. Scale bars, 10 µm. Anterior is to the
left.
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These four classes of "unc-4-motor neurons" (DA, VA, VC,
and SAB) are known to express ChAT and the vesicular acetylcholine transporter (VAChT or UNC-17) (J. Duerr, D. L. Frisby, and J. Rand,
unpublished observations) and are therefore likely to use acetylcholine as an excitatory neurotransmitter. UNC-17 is an integral
membrane component of neurotransmitter vesicles; a substantial fraction
of the ChAT protein is also associated with vesicles in C. elegans (J. Duerr, unpublished observations). Here we show that
the levels of UNC-17 and ChAT as well as that of other
vesicle-associated proteins are regulated by unc-4 in these
cholinergic motor neurons. [unc-4 is also expressed in the
pharyngeal I5 motor neuron and in AVF neurons in the
retrovesicular ganglion (Miller and Niemeyer, 1995), but the effects of
unc-4 mutations on SV proteins were not evaluated in these
cells.]
UNC-4 is required for expression of UNC-17 and ChAT in
unc-4-motor neurons
Antibody staining of wild-type animals for UNC-17 and ChAT
produces a punctate pattern that is characteristic of presynaptic varicosities at en passant synapses. These UNC-17- and ChAT-containing structures are especially well resolved in the ventral and dorsal sublateral nerve cords in the head (Fig.
2) that contain tightly apposed axonal
projections from two classes of cholinergic motor neurons, the SABs and
SAAs (White et al., 1986) (J. Duerr, D. H. Hall, and J. Rand,
unpublished observations). We observed a significant decrease in
UNC-17 and ChAT staining in these processes in the null mutant
unc-4(wd1) (Table 1); ~55%
of the dorsal and ventral head sublateral nerve cords show wild-type
levels of UNC-17 and ChAT staining in unc-4(wd1) animals.
UNC-17 and ChAT staining is either reduced or absent in the remaining
fasicles. [The complete elimination of UNC-17 and ChAT staining in
some cases may mean that both the SAB and SAA axons are affected.
However, we believe that the SAA defect must be indirect because UNC-4
is normally expressed in the SAB motor neurons but not in the SAAs.]
Comparable decreases in UNC-17 and ChAT staining were also observed in
three additional unc-4 alleles including the canonical null
allele unc-4(e120) as well as the missense mutants
e26 and e2321 (Fig. 2, Table 1) (Winnier et al.,
1999). In addition to the decrease in UNC-17 and ChAT antibody
staining, we observed defects in axonal morphology; 10-15% of the
dorsal and ventral head sublateral cords show misplaced, elongated, or
branched processes in unc-4 and unc-37 mutants
(data not shown). These morphologically abnormal SABs (and/or SAAs) also show reduced levels of UNC-17 and ChAT staining.
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Figure 2.
UNC-17 expression in motor neurons is decreased in
unc-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 in
unc-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 the
unc-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.
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UNC-17 and ChAT levels were also evaluated in the VC axons that exit
the ventral nerve cord to innervate vulval muscles (White et al.,
1986). As shown in Figure 2, UNC-17 and ChAT staining of VC
neuromuscular synapses is readily detected in the wild-type animal.
However, in unc-4 mutants, UNC-17 and ChAT staining is substantially reduced in the VC motor neuron presynaptic zones (Fig.
2D, Table 1).
DA and VA motor neurons extend axons into the dorsal and ventral nerve
cords, respectively (Fig. 1) (White et al., 1986). The active zones of
these A-type motor neuronal processes are enmeshed in tightly packed
fascicles and are thus not readily distinguished from the presynaptic
regions of other overlapping motor neurons. To circumvent this problem,
we evaluated UNC-17 and ChAT staining in the presence of a mutation in
the unc-104 kinesin gene (Hall and Hedgecock, 1991). The
unc-104(e1265) mutation blocks anterograde axonal transport
such that SVs accumulate in neuronal cell bodies where they can be
detected by UNC-17 and ChAT antibody staining (Nonet et al., 1993). To
aid in the identification of these unc-4 motor neurons in
the ventral nerve cord, we used an integrated unc-4
promoter-gfp line (wdIs5) to mark the DA and VA
cell nuclei (Pflugrad et al., 1997). In animals containing wild-type
UNC-4 activity, ~80% of the VA and DAs (marked with GFP) show bright
UNC-17 staining (Fig. 2, Table 1). However, in unc-4
mutants, we observed that only ~10% of the VA and DA neurons show
wild-type levels of UNC-17 staining. ChAT staining in the A-type motor
neurons is also reduced in unc-4 mutants (Table 1).
UNC-37 functions with UNC-4 to maintain cholinergic proteins
Other work from this laboratory has shown that the pattern of
synaptic inputs to VA motor neurons (D. Hall, E. German, and D. Miller,
unpublished observations) depends on unc-37 function (Pflugrad et al., 1997; Winnier et al., 1999). Here we show that unc-37 is also required for regulation of cholinergic
protein expression in the VAs and in other unc-4 motor
neurons. UNC-17 and ChAT antibody staining is substantially reduced in
all four types of unc-4 motor neurons in mutants bearing the
hypomorphic allele unc-37(e262) (Table 1) (Miller et al.,
1993). UNC-17 and ChAT staining was not detectably altered in other
classes of cholinergic motor neurons (DB, VB, and AS) in the ventral
nerve cord (data not shown), indicating that this UNC-37 missense
mutation specifically perturbs the regulation of these neurotransmitter
vesicle-associated proteins in unc-4 motor neurons.
UNC-4 and UNC-37 regulate the levels of other
vesicular proteins
UNC-17 is an integral vesicular membrane protein (Alfonso et al.,
1993). Although ChAT is a cytosolic enzyme, a significant fraction of
ChAT protein is associated with the vesicles (Duerr, unpublished
observations). Because these two vesicular proteins are downregulated
in unc-4 and unc-37 animals, we wondered whether other vesicular proteins are also affected in these mutants. We used
specific antibodies to assess the effects of unc-4 and
unc-37 activity on the levels of three additional proteins
that are associated with synaptic vesicles: (1) Synaptotagmin, an
integral vesicular protein proposed to be involved in calcium-mediated
SV release and vesicle endocytosis (Nonet et al., 1993), (2)
Synaptobrevin, an integral vesicular protein that is a component of the
SNARE complex (Nonet et al., 1998), and (3) RAB-3, a GTPase that
associates with vesicles before fusion (Nonet et al., 1997). In all
three cases, the levels of antibody staining were substantially reduced in DA and VA motor neurons in unc-4 and unc-37
mutants (Fig. 3). Thus, our results show
that five different vesicular or vesicle-associated proteins (UNC-17,
ChAT, Synaptotagmin, Synaptobrevin, and RAB-3) are downregulated in
animals with loss-of-function mutations in either the unc-4
or unc-37 genes.
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Figure 3.
Vesicular protein levels depend on
unc-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- and
unc-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. All
unc-4 and unc-37 mutants in these
experiments are loss-of-function alleles.
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Nonvesicular proteins are not affected in unc-4 and
unc-37 mutants
To determine whether unc-4 and unc-37 affect
all proteins involved in exocytosis or whether this effect is
restricted to vesicular proteins, we examined levels of Syntaxin (Ogawa
et al., 1998; Saifee et al., 1998), a neuronal membrane component of
the SNARE complex, as well as that of UNC-18 (Hosono et al., 1992;
Geppert et al., 1994), a cytoplasmic protein implicated in regulating the docking of vesicles via its association with Syntaxin. Expression levels of these proteins were scored in the VC axonal projections and
in the SAB-containing dorsal and ventral sublateral nerve cords in
the head. UNC-18 is readily detected in axonal projections of the VC
motor neurons in wild-type as well as in unc-4 and
unc-37 animals. This finding stands in contrast to our
observation that UNC-17 expression in the VCs is only seen in wild-type
animals (Fig. 4, Table
2).
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Figure 4.
UNC-18 expression is not altered in
unc-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.
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Although Syntaxin expression is not detectable in the VCs, we did
observe high levels of Syntaxin as well as UNC-18 staining in the head
sublaterals in both unc-4 and unc-37 mutants
(Table 2). In this case, it is possible that at least some of the
Syntaxin and UNC-18 staining in the head sublaterals may be caused by
the SAA axons that normally fasiculate with SAB processes. The
differential effects of unc-4 and unc-37
mutations on vesicular proteins are clearly seen in SAB (or SAA) axons
with branching or guidance defects that stain for Syntaxin and UNC-18
but that do not show expression of either RAB-3 or UNC-17 (data not
shown). These results indicate that specific synaptic membrane and
docking regulatory proteins are expressed at wild-type levels in
unc-4 and unc-37 mutants whereas vesicular
proteins are downregulated.
We also assessed the potential effects of the unc-4
mutations on UNC-11, a component of the endocytic machinery. The
C. elegans unc-11 gene encodes a homolog of the clathrin
adaptor protein AP180 (Nonet et al., 1999). In the missense allele
unc-11(n2954), the UNC-11 protein is restricted to motor
neuron cell bodies and does not accumulate at presynaptic termini. This
property of the mutant protein was used to score UNC-11 expression in
DA and VA motor neurons in the ventral nerve cord. Approximately 90%
of ventral cord motor neurons including the DAs and VAs stain positive for UNC-11 in the unc-11 single mutant as well as in the
unc-4;unc-11 double mutant (data not shown). Thus, the
unc-4 mutation does not lower the levels of UNC-11 protein.
This conclusion is also consistent with electron micrographs of VA
motor neuron presynaptic zones that do not show the characteristically
enlarged vesicular structures that are seen in unc-11
mutants (data not shown) (Nonet et al., 1999).
Neurotransmitter vesicles are decreased in
unc-4 mutants
EM reconstruction of unc-4(e120) has revealed that the
VA motor neurons receive incorrect inputs but are otherwise
morphologically wild-type with anteriorly directed axons that make
normal-looking neuromuscular synapses (White et al., 1992). The axonal
diameters of VA motor neurons are similar to those of adjacent VB motor neurons (data not shown). We have now reexamined these EM data to
quantitate the number of vesicles in the VA motor neurons. In wild-type
animals, the average number of vesicles in a section that contains a VA
motor neuron PSD is comparable with the average number of PSD-adjacent
vesicles in VB motor neurons (~40) (Fig. 5) (White et al., 1986). In contrast,
vesicle number in the active zones of VAs is 40% lower than that of
the VBs in unc-4(e120) animals (White et al., 1992). This
difference is statistically significant (see Materials and Methods).
Vesicle numbers within the intersynaptic regions of VAs and VBs are not
significantly different between wild-type and unc-4 animals
(data not shown). This finding is important because it excludes the
possibility that the reduced number of vesicles that we observe at the
presynaptic zones of VA motor neurons in unc-4 mutants is
caused by the redistribution of vesicles within the intersynaptic
regions as is the case in rab-3 mutants (Nonet et al.,
1997). Therefore, we conclude that there are fewer vesicles in the VA
motor neurons in unc-4 mutants and that this reduction is
correlated with the reduced levels of vesicular protein antibody
staining that we have observed in the light microscope.
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Figure 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 and
unc-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 in
unc-4(e120) is significantly different from that of the
VBs (*p < 0.005; **p  0.001).
Error bars indicate SD.
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Reduced vesicular protein staining is correlated with functional
defects in unc-4 neurons
The ventral nerve cord of the newborn L1 larva contains a simple
motor neuron circuit comprised of two classes of excitatory, cholinergic motor neurons (DA and DB) and one class of inhibitory, GABAergic motor neuron (DD) (White et al., 1986; McIntire et al., 1993). [VA motor neurons are not added to the backward movement circuit until the L1-L2 molt (Sulston and Horvitz, 1977)]. Our results showing that SV levels are drastically reduced in the DA motor
neurons in unc-4 and unc-37 mutants predict that
newly hatched L1 larvae should show impaired movement because of the lowered neurotransmitter signaling capacity of ~50% of the
excitatory ventral cord motor neurons in these animals. We used a
liquid "thrashing" assay (Miller et al., 1996) to confirm that
unc-4 and unc-37 L1 larvae are substantially less
active than are wild type (Fig. 6). The
simplest interpretation of this effect is that the excitatory function
of the DA motor neurons is compromised by unc-4 and
unc-37 mutations. EM reconstruction of DA motor neurons in
the adult indicates that these cells are not miswired in
unc-4 mutants and appear otherwise morphologically normal
(White et al., 1992). Therefore, we attribute the movement defect of
unc-4 L1 larvae to the observed reduction in
neurotransmitter vesicles in these cells.
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Figure 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 an
asterisk. Results of experiments performed with
unc-37(wd17) strains are denoted with
gray shading.
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Although the SAB motor neurons make clearly visible synapses adjacent
to muscles in the head, their role in locomotion has not been defined
(J. Duerr, D. H. Hall, and J. Rand, unpublished observations).
Recently, Zhao and Nonet (2000) reported that mutants with reduced
levels of synaptic transmission show SAB axonal sprouting or branching
defects. We have also observed this phenomenon in a significant number
(10-15%) of SAB axons of unc-4 and unc-37 mutants. Furthermore, of the SAB axons that do exhibit sprouting defects, most also show reduced levels of vesicular protein expression (data not shown). Therefore, our results are consistent with the model
in which the lowered levels of neurotransmitter vesicles in
unc-4 and unc-37 mutants result in a significant
reduction in SAB synaptic activity.
The VC motor neurons innervate the vulval muscles to modulate
egg-laying behavior (White et al., 1986; Waggoner et al., 1998). We
have observed that unc-4 and unc-37 mutants are
mildly resistant to serotonin-induced egg laying (Trent et al., 1983)
and also show significantly smaller brood sizes than are seen in wild
type (data not shown). These mutant phenotypes could be indicative of
VC dysfunction in unc-4 and unc-37 mutants.
The VA motor neurons arise after hatching in the first larval stage and
function in the backward movement circuit in larvae and adults (Sulston
and Horvitz, 1977; Chalfie et al., 1985). That VA motor neuron activity
is affected in unc-4 and unc-37 mutants is
evident in the striking backward movement defects that these animals
display (White et al., 1992). The inability of unc-4 mutants
to crawl backward has been attributed to a miswiring defect in which VA
motor neurons receive synaptic inputs from inappropriate command
interneurons (White et al., 1992). It is also possible that the
depletion of neurotransmitter vesicles in the VAs by unc-4
and unc-37 mutations could contribute to this locomotory defect (see Discussion).
UNC-4- and UNC-37-dependent transcriptional repressor function is
required to maintain normal levels of vesicular proteins
Having established that neurotransmitter proteins and the vesicles
into which they assemble are substantially reduced in specific motor
neurons in unc-4 and unc-37 mutants, we next
performed experiments to address the molecular mechanism of this effect.
Missense mutations in the UNC-4 engrailed-like repressor domain (eh1)
have been shown to inactivate UNC-4 repressor activity and to perturb
interactions with UNC-37 (Winnier et al., 1999). We tested two of
these eh1 mutations, e2321 and e26, and
discovered that both result in reduced levels of UNC-17 and ChAT
staining in unc-4 motor neurons (Table 1). Furthermore,
unc-37(wd17), an allele-specific Unc-4 suppressor mutation
in the UNC-37 WD repeat domain (Miller et al., 1993), restores
physical interaction with the e2321-mutant UNC-4 protein and
elevates levels of UNC-17 and ChAT staining in e2321-mutant
animals (Table 1) (Winnier et al., 1999). However, UNC-17 and ChAT
staining is not restored in the null allele unc-4(e120),
which is also not suppressible by the unc-37(wd17) mutation
(Winnier et al., 1999). Thus, we conclude that UNC-4 regulation of
UNC-17 and ChAT protein levels depends on physical interactions with an
intact UNC-37/Groucho protein.
The e2321-mutant animals also show reduced thrashing rates
as L1 larvae (Fig. 6). The unc-37(wd17) mutation suppresses
this effect for the e2321 mutation but not for the
nonsuppressible unc-4(e120) null allele. The concomitant
effects of the unc-37 suppressor mutation on vesicular
protein expression and on L1 motility substantiate a model in which DA
motor neuron function depends on UNC-4 interactions with UNC-37.
These results are consistent with a model in which the UNC-4-UNC-37
complex represses transcription of a downstream gene that in turn
exerts a negative effect on synaptic vesicle levels. Experiments described in the next section indicate that this UNC-4-UNC-37 target
gene (gene x) downregulates SV protein levels via a
post-transcriptional mechanism (see Fig. 9).
Vesicular proteins are regulated at a
post-transcriptional level
unc-17 and cha-1 are arranged in an operon
where they share a common promoter and first untranslated exon (Alfonso
et al., 1994) A 3.2 kb promoter element from this upstream region is
sufficient to drive expression of GFP in virtually all cholinergic
neurons including excitatory motor neurons in the ventral cord (D. Frisby and J. Rand, unpublished observations). Expression of the
unc-17-cha-1 promoter:: gfp reporter
gene in unc-4 motor neurons is not altered by
loss-of-function mutations in either unc-4 or
unc-37 (Fig. 7). Thus, the
unc-17-cha-1 promoter region does not respond to changes in
unc-4 and unc-37 activity. In contrast,
expression of unc-17-cha-1
promoter:: gfp in these neurons does depend on the
cell autonomous activity of the UNC-3 protein that has been shown to
function as a regulator of unc-17-cha-1
transcription (K. L. Lickteig, D. L. Frisby, J. Duerr, D. M. Miller,
and J. Rand, unpublished observations). These findings argue
against a mechanism in which unc-4 and unc-37
mutations affect unc-17 and cha-1
transcription.
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Figure 7.
unc-4 mutations do not affect
unc-17-cha-1 promoter:: gfp
expression. The 3.2 kb unc-17-cha-1
promoter:: 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 and
unc-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. The
arrowhead denotes AS motor neurons that are seen in both
wild-type and unc-4 animals. Asterisks
mark gut autofluorescence. Scale bars, 10 µm.
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To determine whether other vesicular proteins are also regulated at a
post-transcriptional level by unc-4 and unc-37,
we evaluated expression of a GFP-tagged Synaptobrevin
driven by the unc-4 promoter (M. L. Nonet,
1999). This construct
(unc-4p:: SNB-1:: gfp) contains the
full-length Synaptobrevin protein with a C-terminal GFP tag. Expression
of this transgene produces a punctate pattern of GFP staining that is
correlated with the localization of SNB-1:: GFP at the PSDs of
unc-4 motor neurons. This pattern is especially well
resolved in the SAB processes in the head and in VC axons that exit the
nerve cord to innervate vulval muscles (Fig.
8). To determine whether the regulation
of Synaptobrevin by unc-4 and unc-37 is
independent of the Synaptobrevin promoter, we placed the
unc-4p:: SNB-1:: gfp construct in
unc-4- and unc-37-mutant backgrounds. In these
animals, expression of GFP-tagged Synaptobrevin is clearly reduced in
the axonal projections of the VC and SAB motor neurons (Fig. 8). A
similar reduction is seen for UNC-17 and ChAT antibody levels in these
cells in unc-4 and unc-37 mutants (compare Table
1, Fig. 8E). Because unc-4 expression is
not regulated by unc-4 or unc-37 (Miller et al.,
1993; Miller and Niemeyer, 1995), it follows that the decreased levels
of SNB-1:: GFP in these mutants is not a result of
unc-4 or unc-37 regulation of the
unc-4 promoter. Therefore, unc-4 and
unc-37 regulation of Synaptobrevin expression does not occur
via a transcriptional mechanism but must depend on some feature of the
Synaptobrevin-transcribed sequence. This finding parallels the
observation above that UNC-17 and ChAT are also likely to be regulated
by unc-4 and unc-37 at a post-transcriptional
level and therefore favors a model in which all of the affected
vesicular proteins are similarly regulated.
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Figure 8.
GFP-tagged Synaptobrevin is downregulated in
unc-4-mutant motor neurons. The unc-4
promoter 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) and
unc-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.
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Vesicular protein levels are developmentally regulated
by unc-4
We performed temperature-shift experiments with the
temperature-sensitive mutant unc-4(e2322ts) (Miller et al.,
1992) to define the developmental periods in which unc-4 is
required to maintain wild-type levels of the vesicular protein UNC-17
in DA and VA motor neurons. DA motor neurons are generated in the
embryo; VA motor neurons arise after hatching in the late L1 larva
(Sulston and Horvitz, 1977; Sulston et al., 1983). When
e2322ts animals are maintained at the permissive temperature
(16°C) throughout development, both the DA and VA motor neurons
express high levels of UNC-17 protein (Table
3). Growth at the restrictive temperature (25°C) results in substantially lower levels of UNC-17 expression (18% of DAs and 6% of VAs). Temperature-shift experiments have revealed, however, that unc-4 function is specifically
required during the early developmental periods in which DA and VA
motor neurons differentiate but is not necessary in older larvae or adults for maintaining normal levels of UNC-17 protein. For example, exposure of L1 and L2 larvae to 25°C results in UNC-17 staining in
only 30% of the VAs, whereas exposure of older animals (L3 to adult)
does not reduce UNC-17 expression in these cells. DA motor neurons are
similarly sensitive to restrictive temperature. In this case, however,
DAs in both embryos and L1-L2 larvae are affected with only ~20% of
the DAs showing UNC-17 expression; exposure of older animals to
the restrictive temperature has no effect (Table 3).
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DISCUSSION |
unc-4 and unc-37 regulate vesicle
biogenesis or stability
By what mechanism does the unc-4-unc-37
pathway regulate vesicle number in the affected cholinergic motor neurons?
Our results exclude a model in which the decreased number of synaptic
vesicles at the PSD is caused by defective anterograde transport or
localization in unc-4 and unc-37 mutants. First, we showed that SV protein levels, as assayed in the cell soma of DA and
VA motor neurons, are still diminished by unc-4 and unc-37 mutations even when vesicle translocation to the
synapse is blocked by a mutation in the unc-104 kinesin gene
(Hall and Hedgecock, 1991). Second, our analysis of EM serial sections
did not detect any evidence to support a model in which decreased accumulation of SVs at the PSD is caused by their redistribution to
intersynaptic regions as occurs in rab-3 null mutants (data not shown) (Nonet et al., 1997). Although RAB-3 protein levels are
reduced in unc-4 and unc-37 mutants, we assume
that the residual level of RAB-3 expression in the affected motor
neurons of these animals is sufficient for normal SV localization.
In an alternative model, vesicle number could be effectively diminished
by a dramatic increase in exocytosis. If that were the case, mutations
in the unc-13 gene, which is required for vesicle fusion
(Richmond et al., 1999), should prevent SV depletion by this mechanism
in unc-4 and unc-37 mutants. We observed the opposite result, however; the elimination of unc-13 activity
did not restore the SV protein UNC-17 to normal levels in either
unc-4- or unc-37-mutant backgrounds (data not
shown). Thus, our results exclude the possibility that enhanced rates
of vesicle exocytosis account for the reduced levels of SV proteins in
unc-4- and unc-37-mutant motor neurons.
Lastly, our results argue against the possibility that defective
vesicle endocytosis effectively diminishes the steady-state level of
PSD-localized vesicles in unc-4 and unc-37
mutants. unc-4 mutants are not affected in their expression
of UNC-11, a homolog of the endocytic protein AP180 (data
not shown). Although AP180 is not essential for endocytosis, it does
play a role in regulating clathrin coat size; unc-11-mutant
motor neurons show vesicles that are normal in number but with an
enlarged diameter (Nonet et al., 1999). EM analysis of unc-4
mutants does not reveal these large recycling intermediates or other
defects commonly associated with mutations in the endocytic machinery
(i.e., increased axonal diameter, membrane infoldings, collared
vesicles) (Cremona and De Camilli, 1997). In addition, our results
showing that SV levels are decreased even when anterograde transport is
blocked by the unc-104 mutation indicate that nascent SVs
are affected and therefore argue against the possibility that this
reduction is caused by defective endocytosis in unc-4 and
unc-37 mutants.
We favor a model in which unc-4 and unc-37
regulate some aspect of vesicle biogenesis or stability. Genetic
ablation of Synapsin in cultured neurons and in knock-out mice results
in a significant reduction of vesicles and SV proteins but not other
synaptic membrane or cytosolic proteins (Li et al., 1995; Rosahl et
al., 1995; Takei et al., 1995). This effect has been attributed to an
increased rate of vesicle turnover. The striking similarity of this
phenotype to that of unc-4 and unc-37 mutants is
consistent with the formal hypothesis that unc-4 and
unc-37 are indirectly responsible for maintaining Synapsin
protein levels in specific neurons. The recent identification of a
C. elegans Synapsin homolog (Kao et al., 1999) offers the
possibility of experimentally testing this model.
Neuronal function depends on post-transcriptional
regulation of SV protein expression
Because the unc-4 and unc-37 mutations
that reduce SV protein levels are also known to disable the
transcriptional repressor activity of the UNC-4 and UNC-37 proteins, it
seems unlikely that the UNC-4-UNC-37 complex acts as a direct
regulator of SV gene transcription. If that were the case, then SV
protein levels should increase in unc-4 and
unc-37 mutants as opposed to the substantial reduction of
vesicular proteins that we observe in these animals. Thus, we propose
that the UNC-4 and UNC-37 normally function to repress transcription of
an intermediate gene (gene-x) that in turn exerts a
negative effect on SV levels (Fig. 9). In
this model, mutations that disable either unc-4 or
unc-37 result in inappropriate expression of
gene-x that then downregulates SV proteins. Although the
molecular identity of the putative gene-x is unknown, our results indicate that it is likely to act via a post-transcriptional mechanism.
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Figure 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 or
unc-37 loss-of-function mutants, gene-x
is 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 and
unc-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).
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Studies of SV biogenesis in vertebrate neurons have revealed
multiple post-transcriptional levels of control. For example, in
cultured hippocampal neurons, Synaptophysin expression appears to
depend on the rate of mRNA translation, whereas the accumulation of
Synaptotagmin, Synaptobrevin, and Synapsin is linked to increased protein half-life as these neurons develop. The rates of SV assembly and SV protein degradation may be interdependent; SV creation relies on
the available pool of SV proteins, and conversely, precursor proteins
that are not rapidly incorporated into an SV may be degraded (Daly and
Ziff, 1997). Wide variations in SV number and quantal content among
different classes of neurons argue that developmental and homeostatic
control of vesicle-dependent signaling capacity is inherently important
to neuronal physiology (White et al., 1986; Jia et al., 1993;
Merchan-Perez and Liberman, 1996). Tight regulation of SV levels could
be provided by the opposing effects of both positively and negatively
acting pathways. In this instance, the normal function of
unc-4 and unc-37 is to inhibit the activity of a
negatively acting pathway that acts, via a post-transcriptional mechanism, to decrease the levels of SV proteins and the vesicles that
they comprise in specific cholinergic motor neurons.
The negative effect of the unc-4 mutation on cholinergic SVs
also applies to other classes of neurotransmitter vesicles.
Antibody-staining experiments have detected reduced expression of the
neuropeptide FMRFamide (Li and Chalfie, 1990; Schinkmann and Li, 1992)
(C. Li, personal communication) and the vesicular monoamine
transporter in the VC motor neurons (Duerr et al., 1999) (Duerr,
unpublished observations). These observations indicate that
unc-4 and unc-37 may mediate a general event in
the creation or maintenance of all types of neurotransmitter vesicles.
Experiments with a temperature-sensitive unc-4 mutant
indicate that SV levels depend on unc-4 function during a
brief period of early development but do not require unc-4
activity in the adult. This finding may mean that unc-4 and
unc-37 mediate a key event during the differentiation of an
affected motor neuron that then sets the steady-state level of SV
proteins in the mature cell (see below).
Motor neuron dysfunction contributes to the Unc-4
movement defect
Body wall muscle excitation during backward locomotion has been
attributed to the coordinated activities of cholinergic DA and VA motor
neurons and the command interneurons (AVA, AVD, and AVE) that drive
them (Chalfie et al., 1985). The striking backward movement defect
shown by unc-4 and unc-37 loss-of-function
mutants is correlated with the loss of normal inputs to VA motor
neurons. These synapses are replaced with gap junctions from a command interneuron (AVB) that usually mediates forward locomotion (White et
al., 1992) (Hall, German, and Miller, unpublished observations). Recent
results suggest that the simple elimination of synaptic inputs to the
VAs may be insufficient to account for the total abrogation of backward
movement in unc-4 mutants; genetic ablation of the
"backward command interneurons" (AVA, AVD, and AVE) retards but
does not block reverse locomotion (Zheng et al., 1999). Our finding
that the neurotransmitter signaling capacity of VA motor neurons is
also reduced in unc-4 mutants may provide an explanation for
the enhanced severity of the Unc-4 backward movement defect.
Does the vesicular signaling capacity of VA motor neurons specify
presynaptic inputs or vice versa?
Experiments with a temperature-sensitive unc-4 mutant
indicate that both the input and output defects of VA motor neurons are
caused by the loss of unc-4 activity during early larval
development. VA motor neurons are generated during the first larval
stage (L1) (Sulston and Horvitz, 1977). Although the adult pattern of
presynaptic connections is established before the L1-L2 molt (J. White, personal communication), additional motor neuron inputs
are probably added during subsequent larval development to accompany
dramatic increases in neuronal size (Rongo and Kaplan, 1999). If
unc-4 activity is effectively turned off during this
critical period (L2 to mid-L3) by exposing the unc-4ts
allele to a restrictive temperature, then adult animals show a strong
backward movement defect (Miller et al., 1992) as well as a reduction
in synaptic vesicles in the VAs. The temporal correlation of the
miswiring event and the reduction in VA signaling capacity is
suggestive of a causal relationship between these defects.
In one model, the change in synaptic inputs triggers the depletion of
synaptic vesicles in the VAs. However, an identical mechanism could not
account for the loss of synaptic vesicles in the DAs because these
motor neurons are not miswired in unc-4 mutants. Perhaps the
unc-4 mutation induces transient miswiring of the DAs (and
SAB and VC motor neurons), and this brief loss of normal inputs is
sufficient to induce the synaptic vesicle defect.
It is also possible that the opposite mechanism applies. The loss of
vesicular proteins in the VA motor neurons results in an altered
pattern of presynaptic inputs. This model supposes that either the
vesicular proteins or the signaling activity that they provide may
define presynaptic specificity. Ample evidence exists for models in
which both the strength and selectivity of presynaptic connections can
be influenced by retrograde signals from the postsynaptic partner. The
mechanisms of these effects, however, are primarily unknown
(Fitzsimonds and Poo, 1998). Recent experiments have shown that
N-ethylmaleimide-sensitive factor (NSF), a cytosolic
protein involved in vesicular fusion, may be involved in targeting
glutamate receptors to the postsynaptic membrane (Nishimune et al.,
1998; Osten et al., 1998; Song et al., 1998). In addition, the
application of inhibitors of NSF activity or cleavage of the SNARE
protein Synaptobrevin by microinjection or cell autonomous expression
of tetanus toxin can reduce the strength of synaptic inputs to the
affected neurons (Lledo et al., 1998; Baines et al., 1999). These
studies indicate that SV fusion may be important for the correct
localization of receptors at the postsynaptic membrane in addition to
providing neurotransmitter signaling capacity at the presynaptic
density. It is conceivable that a similar effect on the localization of
key receptor or marker proteins in the VA postsynaptic membrane could
effectively specify presynaptic inputs. This model predicts that VA
motor neurons should adopt the VB pattern of synaptic inputs in mutants
in which SV accumulation or activity is reduced (Jorgensen et al.,
1995). This model also assumes, of course, that the loss of SV activity in other classes of motor neurons (e.g., the DAs) is not sufficient to
alter presynaptic inputs.
Lastly, it is conceivable that the loss of synaptic vesicles and the
rewiring of VA motor neurons are independently induced by the
unc-4 mutation and have no mechanistic relationship other than the common link to unc-4 activity. Perhaps the most
effective way to resolve this question is to identify the
unc-4 target genes that mediate these events.
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FOOTNOTES |
Received June 20, 2000; revised Oct. 16, 2000; accepted Nov. 28, 2000.
This work was supported by National Institutes of Health Grants MH12260
(K.M.L.), NS26115 (D.M.M.), GM38679 (J.B.R.), and NCRR12596 (D.H.H.)
and OCAST Grant HN3-023 (J.S.D.). We thank R. Blakely and
members of the D. M. Miller and D. Greenstein laboratories for
helpful discussions; M. Nonet for antibodies against RAB-3, Synaptobrevin, and Syntaxin and for the unc-4
promoter-SNB-1::GFP plasmid; A. Alfonso for the
unc-11(n2954) strain and UNC-11 antibody; John White for
wild-type and unc-4(e120) electron micrographs; Tylon
Stephney for scanning EM prints; and Chris Li for communicating unpublished data.
Correspondence should be addressed to Dr. David M. Miller, III, C2310
Medical Center North, 1161 21st Avenue South, Vanderbilt University,
Nashville, TN 37232. E-mail: david.miller{at}mcmail.vanderbilt.edu.
| |
REFERENCES |
-
Alfonso A,
Grundahl K,
Duerr JS,
Han HP,
Rand JB
(1993)
The Caenorhabditis elegans unc-17 gene: a putative vesicular acetylcholine transporter.
Science
261:617-619[Abstract/Free Full Text].
-
Alfonso A,
Grundahl K,
McManus JR,
Asbury JM,
Rand JB
(1994)
Alternative splicing leads to two cholinergic proteins in Caenorhabditis elegans.
J Mol Biol
241:627-630[ISI][Medline].
-
Baines RA,
Robinson SG,
Fujioka M,
Jaynes JB,
Bate M
(1999)
Postsynaptic expression of tetanus toxin light chain blocks synaptogenesis in Drosophila.
Curr Biol
9:1267-1270[ISI][Medline].
-
Bergmann M,
Lahr G,
Mayerhofer A,
Gratzl M
(1991)
Expression of synaptophysin during the prenatal development of the rat spinal cord: correlation with basic differentiation processes of neurons.
Neuroscience
42:569-582[ISI][Medline].
-
Brenner S
(1974)
The gen
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ABSTRACT
INTRODUCTION
