Download UNC-115, a Conserved Protein with Predicted LIM and Actin

Neuron, Vol. 21, 385–392, August, 1998, Copyright 1998 by Cell Press
UNC-115, a Conserved Protein with
Predicted LIM and Actin-Binding Domains,
Mediates Axon Guidance in C. elegans
Erik A. Lundquist,*‡ Robert K. Herman,†
Jocelyn E. Shaw,† and Cornelia I. Bargmann*
* Howard Hughes Medical Institute
Department of Anatomy
The University of California
San Francisco, California 94143-0452
† Department of Genetics and Cell Biology
University of Minnesota
St. Paul, Minnesota 55108
Summary
Axon guidance receptors modulate the growth cone
cytoskeleton through signaling pathways that are not
well understood. Here, we describe the C. elegans
unc-115 gene, which encodes a candidate cytoskeletal linker protein that acts in axon guidance. unc-115
mutants have defects in a subset of axons, particularly
as the affected axons change environments during
outgrowth. The unc-115 gene encodes a putative actin-binding protein that is similar to the human actinbinding protein abLIM/limatin; it has a villin headpiece
domain and three LIM domains that could mediate
protein interactions. unc-115 is expressed in neurons
during their development and is required cell-autonomously in certain neurons for normal axon guidance.
We propose that UNC-115 modulates the growth cone
actin cytoskeleton in response to signals received by
growth cone receptors.
Introduction
During axon guidance, activation of guidance receptors
on the growth cone alters actin cytoskeleton dynamics
and modulates growth cone migration (Lin et al., 1994).
The growth cone modulates actin assembly and disassembly during the extension and retraction of multiple
actin-based filipodia at its leading edge. However, the
signaling pathways utilized by most axon guidance receptors are unknown. Known catalytic domains or signaling motifs are absent from the cytoplasmic domains
of the UNC-6/netrin receptors UNC-40/DCC/frazzled
and UNC-5 (Hedgecock et al., 1990; Leung-Hagesteijn
et al., 1992; Chan et al., 1996; Keino-Masu et al., 1996),
as well as the semaphorin III receptor neuropilin (Takagi
et al., 1991). Receptor protein tyrosine kinases, including
those of the Eph family, and receptor tyrosine phosphatases are implicated in axon guidance (Desai et al., 1997;
Orioli and Klein, 1997). However, the potential substrates
for tyrosine phosphorylation are largely unknown, and,
in the case of the Eph family member Nuk, kinase activity
is not required for guidance of the mouse anterior commissural axons (Henkemeyer et al., 1996).
How are the cytoplasmic domains of guidance receptors coupled to the cytoskeleton? Candidate molecules
downstream of guidance receptors include members of
‡ To whom correspondence should be addressed.
the Rac/Rho/Cdc42 family of small GTPases, which
have been implicated in actin cytoskeleton modulation
in response to extracellular signals (Ridley and Hall,
1992; Ridley et al., 1992). Constitutively active forms of
Cdc42, Rac, and Rho can alter actin cytoskeleton dynamics and cell morphology of cultured cells in distinct
ways (Tapon and Hall, 1997). Some of these changes,
such as filipodial and lamellipodial extension, are reminiscent of the behavior of neuronal growth cones, and,
indeed, Rac/Rho/Cdc42 family GTPases can affect axon
outgrowth. Gain-of-function Rac/Cdc42 proteins can
disrupt guidance of Drosophila melanogaster and C.
elegans axons (Luo et al., 1994; Zipkin et al., 1997), and
mutation of a Caenorhabditis elegans Rac GTP exchange factor (UNC-73) disrupts axon guidance (Steven
et al., 1998). Another type of cytoplasmic protein with
a role in axon outgrowth is encoded by the Drosophila
enabled (ena) gene (Gertler et al., 1995); the mouse Ena
protein can affect actin dynamics in cultured cells (Gertler et al., 1996). Small GTPases have been implicated in
many cellular processes (Chant and Stowers, 1995), and
both the GTPases and ena molecules affect outgrowth
of many or most axons in Drosophila. These observations raise the possibility that other factors in the growth
cone confer specific properties on particular axon guidance events.
Here, we describe the characterization of the C. elegans unc-115 gene, which encodes a potential actinbinding protein involved in axon guidance. Mutations in
unc-115 cause specific defects in axon guidance and
do not affect axon outgrowth per se. UNC-115 is similar
to the human actin-binding protein abLIM/limatin (Kim
et al., 1997; Roof et al., 1997), a candidate tumor suppressor gene. Cytoskeletal LIM domain proteins are
widespread and highly conserved among animals, but
their functions are not well understood; here, we implicate a protein of this class in axon guidance. We propose
that UNC-115 exerts its effect on axon morphogenesis
by influencing the response of the actin cytoskeleton to
guidance signals.
Results
unc-115 Encodes a Putative Actin-Binding Protein
The mutation unc-115(e2225) was identified by D. ThierryMieg based on its uncoordinated phenotype. We identified a new allele, unc-115(mn481), that appeared spontaneously in a strain with active Tc1 transposable elements
(Mori et al., 1988). Two additional unc-115 alleles (ky274
and ky275) were identified in a noncomplementation
screen against unc-115(mn481) following EMS mutagenesis (see Experimental Procedures).
unc-115 was cloned by identifying the Tc1 transposon
in the unc-115(mn481) allele, which fell within the predicted gene F09B9.2. Several lines of evidence indicate
that this gene corresponds to unc-115. First, a minimal
clone containing the F09B9.2 gene rescued the uncoordinated phenotype and the phasmid axon phenotypes
(see below) of all four unc-115 mutants. Second, each
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Figure 1. UNC-115 Is Similar to Vertebrate
Actin-Binding Proteins
(A) Exon/intron structure of the unc-115 gene
deduced from cDNA sequence and genomic
sequence. The cosmid F09B9 was sequenced
by the C. elegans Genome Sequencing Consortium (GenBank accession #Z49887). Boxes
represent exons, lines represent introns, and
black boxes represent coding region. SL1 refers to the site of addition of the trans-spliced
leader sequence, ATG marks the site of the
first in-frame methionine, and TAA is the stop
codon. The bottom line shows the 59 end of
cDNA yk97f4.5. The upper lines represent the
classes of SL1-spliced transcripts generated
by RT-PCR. The positions of the four unc115 mutations are indicated. The positions of
the GFP fusions are indicated below the gene
structure.
(B) Conceptual translation of the longest SL1spliced unc-115 cDNA. The LIM domains, villin headpiece domain, and region of similarity
between UNC-115, abLIM, and dematin (UAD
region) are underlined. The first in-frame methionine residue in the shorter unc-115 transcripts (residue 170) is highlighted.
(C) Structure of UNC-115 polypeptides encoded by the long and short unc-115 transcripts.
(D) Alignments of UNC-115 with other proteins. The three UNC-115 LIM domains are
shown aligned with their most similar counterparts from abLIM (GenBank accession
#AF005654). Identity is highlighted and quantified to the right of the alignment, and a consensus LIM domain sequence is below. An
alignment of the villin headpiece domain regions of UNC-115, abLIM, human dematin
(GenBank accession #U28389), Drosophila
quail (GenBank accession #U10070), and human villin (GenBank accession #X12901) is
shown. Identity between three or more polypeptides is highlighted. Stop codons are denoted by an asterisk. The region containing
basic residues important for the function of
the VHD from human villin is underlined. Also
shown is an alignment of the UAD region of
identity shared by UNC-115, abLIM, and dematin.
of the four unc-115 mutations had a lesion within the
F09B9.2 gene (Figure 1A): mn481 is an insertion of a
Tc1 element in the fifth exon, e2225 is an insertion of a
Tc4 transposable element (Yuan et al., 1991) at the exon
6/intron 6 boundary, ky274 is a TGG(trp) to TGA(opal)
alteration at amino acid residue 450, and ky275 is a
TGG(trp) to TAG(amber) alteration at amino acid residue 489.
cDNA clones of the unc-115 locus defined a family of
related unc-115 transcripts that differ by alternate 59
exons (Figure 1A). Each of the four unc-115 mutations
is predicted to affect all unc-115 transcript classes. The
longest SL1-spliced unc-115 cDNA could encode a 639
amino acid polypeptide (UNC-115) with three regions at
the N terminus similar to the LIM domain and a region
at the C terminus similar to the actin-binding villin headpiece domain (VHD) (Figures 1B–1D). The two shorter
SL1-spliced transcripts are predicted to encode a 470
amino acid polypeptide missing the first two LIM domains (Figures 1B and 1C). The domain organization of
UNC-115 is similar to that of the recently described
abLIM protein (also known as limatin) from humans (Roof
et al., 1997), which is a candidate tumor suppressor gene
(Kim et al., 1997). The largest known abLIM molecule has
four LIM domains at the N terminus and a VHD at the C
terminus; other abLIM isoforms have fewer LIM domains
but include the VHD. LIM domains are zinc-binding motifs, similar in structure to the zinc-finger, that mediate
protein–protein interactions (Dawid et al., 1995). Figure
1D shows an alignment of UNC-115 LIM domains with
those of abLIM. The abLIM and UNC-115 LIM domains
share extensive similarity beyond the LIM consensus
residues, suggesting that the LIM domains have functions that are conserved across the two proteins.
The villin headpiece domains from villin (Friederich et
al., 1992), abLIM (Roof et al., 1997), and dematin (Rana
et al., 1993) can bind actin filaments, and the human
villin VHD can induce the formation of actin filaments
from G-actin. Figure 1D shows an alignment of UNC115 with these VHDs and the VHD of the Drosophila quail
Cytoskeletal Axon Guidance Protein UNC-115
387
gene product, which is involved in actin organization
in the developing egg chamber (Mahajan-Miklos and
Cooley, 1994). The UNC-115 VHD is most similar to the
VHDs of abLIM and dematin and includes the basic
residues that are implicated in actin–villin interactions.
We also identified a conserved motif of unknown function in the middle region of UNC-115, abLIM, and dematin, that contains the residues PAAxxPDP (Figure 1D),
which we call the UAD region.
unc-115 Null Mutants Have Specific Axon
Guidance Defects
All unc-115 mutant phenotypes are recessive, suggesting that the mutations cause loss of unc-115 function. All four unc-115 mutants display uncoordinated
locomotion and are viable and fertile as homozygotes
and viable and fertile in trans to a deficiency that deletes
the locus (nDf19). ky274 and ky275 are probably null
alleles: both cause premature termination of the UNC115 protein, and their phenotypes are not enhanced in
trans to nDf19.
The morphologies of several axons were defective in
unc-115 mutants (Table 1; Figure 2). unc-115 mutants
have a defect in dorsal outgrowth of the VD and DD
motor neurons that may contribute to their uncoordinated movement. In wild-type, these neurons have ventral cell bodies and a branched process that grows anteriorly and then dorsally to the dorsal nerve cord, where
it branches again and grows both anteriorly and posteriorly (Figures 2A and 2B) (White et al., 1986). In unc-115
mutants, the anterior axon branches were normal, but
the dorsal branches stopped short in the lateral body
region and then turned posteriorly (Figure 2C; Table 1).
All VD and DD axons were equally affected except for
DD1 and VD2, which are included in a commissural fascicle with the DA1 and DB2 axons.
unc-115 mutants also exhibited premature termination and misrouting of the sublateral nerves. The sublateral SIADL, SMBDL, and SMDDR neurons have cell
bodies in the head and posteriorly directed processes
that jog toward the lateral midline near the vulva (Figures
2D and 2E) (White et al., 1986). In unc-115 mutants, these
axons terminated before reaching the vulva, sometimes
ending in ectopic branches and misguided axons (Figure
2F; Table 1).
The phasmid sensory neurons in the tail also exhibit
premature termination. The phasmid neurons have axons that extend first ventrally and then anteriorly in the
nerve cord and dendrites that extend posteriorly (Figure
2G) (White et al., 1986). In unc-115 mutants, the phasmid
axons terminated prematurely in the ventral cord (Figure
2H; Table 1).
Finally, we observed subtle fasciculation defects in
the ventral nerve cord of unc-115 mutants. The C. elegans ventral nerve cord (VNC) consists of a large right
bundle and a small left bundle. Wightman et al. (1997)
previously showed the HSNL and AVKR neurons, which
normally extend in the left VNC, extend in the right VNC
in unc-115 mutants. Additionally, we have found that in
unc-115 mutants the PVPR and PVQL axons of the left
VNC cross to the right VNC near the end of their trajectories (Table 1). Thus, all four neurons of the left VNC
exhibit crossover defects. In addition, axons on the right
VNC exhibit weak fasciculation defects in unc-115 mutants (data not shown), possibly contributing to the Unc
phenotype.
Many axons were normal in unc-115 mutants. For
example, the DA motor axons have dorsal trajectories
similar to the VDs and DDs (White et al., 1986) but were
unaffected by unc-115 mutations (data not shown), indicating that some dorsal guidance cues are present in
unc-115 animals. Similarly, the anterior–posterior outgrowth of the CAN, ALM, and PLM lateral axons was
normal in unc-115 mutants, as were the long-range cell
migrations of the HSN and CAN neurons and the Q
neuroblasts. All neuron cell bodies were located in approximately normal positions, and all nerve bundles
were present in their correct positions, including the
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Figure 2. Axon Guidance Defects in unc-115
Mutants
In all micrographs, dorsal is up and anterior
is to the left. Scale bars represent 5 mm.
(A) Diagram of an adult C. elegans showing
all VD and DD ventral-to-dorsal commissural
axons between the ventral and dorsal nerve
cords. The cell bodies of the VD and DD motor
neurons (open circles) sit along the ventral
nerve cord.
(B and C) Confocal images of VD and DD
commissural motor axons in unc-115(1) (B)
and unc-115(ky275) (C) animals. Commissural axons are marked by arrowheads, dorsal and ventral nerve cords are indicated by
arrows, and the large arrowhead in (C) marks
where the VD4 axon ends in an abnormal varicosity.
(D) A diagram of an adult C. elegans showing
the left dorsal and ventral sublateral nerves.
Cell bodies of the left SIA, SMB, and SMD
neurons are indicated by open circles. The
vertical line near the middle of the animal represents the vulva.
(E and F) Epifluorescence images of the SIA,
SMB, and SMD axons in the left dorsal sublateral nerves of unc-115(1) (E) and unc115(ky275) (F) animals. The nerves are marked
by small arrowheads, and the position of the
vulva is indicated by a large arrowhead. The
arrow in (F) marks where axons in the nerve
terminate or become misrouted.
(G and H) Phasmid neurons in unc-115(1) (G)
and unc-115(ky275) (H) animals. The axons
are marked with arrows, the bright spots posterior to the axons are the cell bodies, and
the dendrites extend posteriorly from the cell
bodies.
nerve ring, the ventral and dorsal nerve cords, and the
dendritic bundles in the nose and tail of the animal.
plaques along the excretory canals (Figure 3D) as well
as plaques at the junctions of epidermal cells (Figure 3E).
unc-115 Is Expressed in Neurons and Epidermis
and Localizes to the Cell Cortex
Cells that could express unc-115 were identified using
a full-length UNC-115::GFP fusion gene that rescued
unc-115 mutant defects (Figure 1A). The earliest detectable expression of UNC-115::GFP was in neurons and
epidermis at about 300 min postfertilization, when the
embryo begins to elongate, and axons begin to grow
(Wood, 1988) (Figure 3A). UNC-115::GFP and the shorter
fusion GFPS was expressed in most or all neurons
throughout development (Figure 3B). UNC-115::GFP expression was also observed in nonneuronal cells, including the epidermal syncytium hyp7, the head and tail
epidermal cells, the excretory canal cell, the pharynx,
and the developing vulva, but not in the lateral epidermal
seam cells or the ventral epidermal P cells. We could
detect no defects in nonneuronal tissues in unc-115
mutants.
UNC-115::GFP protein was present uniformly in neuronal cell bodies and processes and was excluded from
nuclei (Figure 3C). The protein was also present at high
levels in the growth cones of developing axons as they
extended to their targets and in cell-cortex-associated
unc-115 Function Is Required in Neurons
The widespread expression of unc-115 suggested that
unc-115 might act in the epidermis or in neurons to
promote axon guidance. To distinguish between these
possibilities, we analyzed unc-115 function in genetic
mosaics (Herman, 1995). unc-115 mosaics were isolated
from unc-115(mn481) lin-15 animals transgenic for an
extrachromosomal array of DNA, kyEx209, that contains
the full-length unc-115::gfp fusion gene and lin-15(1)
DNA. The multivulval phenotype of lin-15 is due to absence of lin-15 activity in epidermal cells (Herman and
Hedgecock, 1990). We identified Unc non-Muv mosaic
animals and deduced the point of array loss by scoring
cells for the expression of UNC-115::GFP, which we
assume is cell-autonomous (Figure 4A). Because arrays
of cloned genes often show multiple losses in a single
animal, we only interpret the effects of mosaic losses
that were observed two or more times.
Losses of the array in either the ABpr or ABpl lineages
resulted in an Unc non-Muv phenotype (Figure 4). All
of the losses that caused Unc phenotypes occured in
lineages that give rise to many motor neurons. Many of
these lineages also give rise to adjacent epidermis, but
Cytoskeletal Axon Guidance Protein UNC-115
389
Figure 3. unc-115 Is Expressed in Neurons
and Epidermis and Localizes to the Cell
Cortex
In all panels, dorsal is up and anterior is to
the left. All animals are wild-type.
(A) A 1.5-fold embryo showing GFPS expression in neurons, epidermis, and epidermiblasts in the anterior, posterior, and ventral
regions (arrowheads).
(B) A late L1 larva showing GFPS expression
in the entire nervous system, including all
neurons in the head and tail (arrows) and
along the ventral cord (arrowheads).
(C–E) Micrographs of animals harboring a
functional GFPFL transgene. (C) A left ventral–lateral confocal image of neurons and
processes near the left posterior lateral ganglion of an adult animal is shown. The cell
bodies of PVM, SDQL, PVDL, and PDEL are
labeled. Note exclusion of UNC-115::GFP
from nuclei. Axons and axon fascicles are labeled: left (L) and right (R) fascicles of the
ventral cord; a commissural process of a ventral cord motor neuron (C); the left sublateral
nerves (SLN); and the canal-associated nerve
(CAN). (D) UNC-115::GFP expression in the
excretory canal of an adult animal. UNC115::GFP accumulates in plaques (arrowheads) approximately 0.3 mm wide that form
two rows down the cortex of the excretory
canal. (E) UNC-115::GFP in the epidermal
syncytium of a newly hatched L1 larva. UNC115::GFP accumulates in plaques at the cortex of the syncytium (arrows) that are 1–3 mm
in width. The lateral epidermal seam cells,
which do not express UNC-115::GFP, are labeled V1–V6. The pharynx shows strong expression of UNC-115::GFP.
five Unc non-Muv mosaic animals had a loss at ABplp,
which gives rise to 10 motor neurons and only two epidermal nuclei in the tail (hyp8/9 and hyp10) that are
unlikely to affect locomotion. Since uncoordinated movement was observed in mosaics with a majority of wildtype epidermal nuclei (all Unc non-Muv animals exhibited epidermal UNC-115::GFP expression), epidermal
unc-115 is not sufficient for normal locomotion. We suggest that unc-115 expression in neurons is required for
coordinated movement.
To examine axon guidance in more detail, the unc115 cDNA (a full-length clone representing the longest
SL1-spliced variant) was placed under the control of
neuron-specific promoters and tested for the ability to
rescue axon guidance defects of unc-115 mutants. An
unc-115 minigene containing a shortened unc-115 promotor with neuron-specific expression was able to rescue the VD and DD axon defects of unc-115(ky275) mutants and partly rescue their Unc phenotype and their
phasmid axon phenotype (Figure 4B). This minigene is
expressed only in a subset of neurons, including the
motor neurons and phasmid neurons, where its expression peaks in the embryo and is low after the L1 larval
stage; no nonneuronal expression of this minigene was
observed.
Further evidence for cell autonomy was obtained by
expression of the unc-115 cDNA::gfp fusion in the phasmid neurons using the promoter of the osm-6 gene.
osm-6 is expressed only in 56 sensory neurons, five of
which are in the tail (the four phasmid neurons and the
PQR neuron) (Collet et al., 1998). The osm-6::unc-115
transgene displayed the same sensory-specific expression profile as osm-6 and partially rescued the phasmid
axon phenotype but not the uncoordinated phenotype
of unc-115(ky275) mutants (Figure 4B). Together with
the mosaic analysis, these results suggest that unc-115
expression in motor neurons or phasmid neurons can
promote proper axon guidance in the absence of surrounding epidermal expression. The partial rescue of
phasmid axons may be due to poor transgene expression or to effects of other neurons or epidermis on phasmid axon extension (Wightman et al., 1996).
Discussion
unc-115 encodes a polypeptide with LIM domains and
a villin headpiece domain that is similar to the human
actin-binding protein abLIM/limatin, a candidate tumor
suppressor gene (Kim et al., 1997; Roof et al., 1997). LIM
domains mediate protein-protein interactions, including
dimerization with other LIM domains as well as heterologous interactions with other motifs (Arber and Caroni,
1996; Wu et al, 1996). The conservation between the
LIM domains of UNC-115 and abLIM, particularly between the third UNC-115 LIM domain and the fourth
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390
Figure 4. unc-115 Is Cell Autonomous
(A) Mosaic loss of unc-115 in early divisions
of the C. elegans embryo. Listed in black below the arrows are the cells that were inspected for UNC-115::GFP expression in the
mosaic animals. Blue boxes indicate the lineages that give rise to motor neurons, and
red bars mark the lineages that give rise to a
majority of epidermal nuclei. A closed circle
next to a cell position indicates the latest discernable point in the lineage that an unc115(mn481) lin-15 mosaic animal had a single
loss of the array containing GFPFL and lin15(1) DNA; the actual loss could have been
later in some cases. Five Unc non-Muv mosaic animals, not shown in the figure, had no
discernable loss, suggesting a late loss in a
lineage where expression could not be scored
using UNC-115::GFP. Losses at ABprp would
be of this class.
(B) Rescue of unc-115 axon phenotypes by
neural-specific expression of the unc-115
cDNA. The percent of wild-type axons (y-axis)
in animals of different genotypes (x-axis) are
shown. The axon phenotypes of the phasmid
axons (black), the DD2 axon (grey) and the
VD4 axon (stippled) are described in Figure 3.
The differences between all unc-115(ky275);
transgene phenotypes and those of unc115(ky275) alone are significant (p , 0.001).
abLIM LIM domain, suggests that they interact with similar protein motifs. UNC-115 is also likely to interact with
the actin cytoskeleton via the villin headpiece domain
(VHD). Although we have not demonstrated this interaction directly, similar domains in human villin (Friederich
et al., 1992), dematin (Rana et al., 1993), and abLIM
(Roof et al., 1997) are known to bind actin filaments,
and key residues involved in actin interactions are conserved in the UNC-115 VHD. LIM domain proteins that
lack apparent actin-binding motifs are known to associate with the actin cytoskeleton through unknown binding
partners. For example, zyxin and paxillin both contain
LIM domains and localize to actin-rich focal adhesions
(Macalma et al., 1996; Turner and Miller, 1994), and some
LIM-only proteins are associated with actin filaments
(Arber and Caroni, 1996). Molecules like UNC-115 and
abLIM may provide a scaffold for assembly of other LIM
proteins onto the cytoskeleton.
unc-115 mutants display axon defects in a variety of
neurons. Guidance and outgrowth in the dorsal-ventral
axis (the VD and DD axons) and in the anterior–posterior
axis (the sublateral nerves and the phasmid axons) are
affected. unc-115 affects the VD and DD axons that
grow on an epidermal substrate as well as many axons
that fasciculate with other neurons. In all cases, unc115 mutant axons can complete some aspects of their
outgrowth normally, such as the ventral extensions of
the phasmid axons and the anterior extensions of the
VD and DD axons in the ventral cord, suggesting that
the axons’ ability to extend is not compromised by unc115 mutation. Indeed, in most cases the affected axons
reach their full length after making guidance errors.
Other C. elegans genes with widespread effects on axon
outgrowth and guidance encode cytoplasmic proteins,
including unc-33 (Li et al., 1992), unc-44 (ankyrin-like)
(Otsuka et al., 1995), unc-51 (serine/threonine kinase)
(Ogura et al., 1994), unc-76 (Bloom and Horvitz, 1997),
and unc-73 (rac/cdc42 GTP exchange factor) (Steven et
al., 1998). All of these genes affect many axons, suggesting a general role in axon guidance and outgrowth,
unlike unc-115, which affects a small number of guidance decisions.
All axons affected by unc-115 mutation have multiple
aspects to their outgrowth, and all change direction or
substrates during their extension. unc-115 defects are
most apparent during these changes. unc-115 mutant
sublateral axons fail just as they are changing their outgrowth direction to a more lateral position, the PVP and
PVQ axons normally change neighbors at the point
where unc-115 defects occur, the phasmid axons in
unc-115 mutants fail when they switch from ventral migration to anterior migration, and the VD and DD axons
Cytoskeletal Axon Guidance Protein UNC-115
391
are defective only in the dorsal aspect of their outgrowth.
unc-115 could be needed to respond to a specific set
of guidance cues, or it could be required more generally
for an axon to switch priorities during outgrowth.
UNC-115 represents a new type of actin-binding protein that is required in developing neurons for proper
axon outgrowth. Although its expression is widespread,
its mutant phenotype is quite specific to certain guidance decisions. To date, no other UNC-115-like molecules have been discovered by the C. elegans Genome
Sequencing Project, arguing against redundancy of unc115 function. Like the rho/rac/cdc42 proteins, UNC-115
is a candidate molecule to link cell signals to changes
in cell shape. We speculate that UNC-115 regulates actin
assembly, disassembly, or actin filament bundling in
response to guidance signals.
Experimental Procedures
unc-115 Genetics
Nematodes were cultured at 208C as described by Brenner (1974).
The wild-type strain was N2. The mutation mn481 arose spontaneously in the mutator strain RW7097, in which the transposable element Tc1 is active in the germline (Mori et al., 1988) and was mapped
to the X chromosome. By Southern blots, we identified a 4.3 kb
Tc1-containing fragment present in the unc-115(mn481) strain that
was absent in N2 and two spontaneous revertant strains. A 2.9 kb
EcoRV–HindIII genomic DNA fragment flanking the Tc1 insertion
was cloned and includes a fragment of the unc-115 gene.
F1 Noncomplementation Screen for New
unc-115 Alleles
N2 males treated with ethyl methanesulfonate were mated to dpy-6
unc-115(mn481) egl-15 hermaphrodites, and F 1 Unc non-Dpy nonEgl hermaphrodite progeny were sought. Among 25,000 F1 progeny
screened, we found two unc-115 mutations, ky274 and ky275. Each
allele was outcrossed to N2 five times. The unc-115 allele referred
to as mn490 in Wightman et al. (1997) was mn481.
Visualization of Axons
Axons were visualized in living animals by expression of the green
fluorescent protein from neuron-specific promotors and by filling of
the amphid and phasmid chemosensory neurons with the fluorescent dye 3,39-dioctadecyloxacarbocyanine perchlorate (Herman
and Hedgecock, 1990). Some animals were fixed in 2% paraformaldehyde for 2 hr to reduce gut autofluorescence. All animals were
scored as young adults.
The following promotor::gfp fusions were used: unc-115 GFPS to
visualize overall nervous system structure, dsp2::gfp (N. L’Etoile and
C. I. B., unpublished data) for the commissural axons of the VD and
DD motor neurons, ZC21.2::gfp (Colbert et al., 1997) for the SIA,
SMB and SMD sublateral axons, odr-2::gfp (J. Chou and C. I. B.,
unpublished data) for the PVP axons, and sra-6::gfp (Troemel et al.,
1995) for the PVQ axons.
Molecular Biology
Standard molecular biology techniques were used. Five genomic
clones were isolated by hybridization of the unc-115 genomic fragment to a C. elegans genomic phage l EMBL4 library (generously
provided by Chris Link). Five unc-115 cDNAs were isolated from a
mixed stage lZAP C. elegans cDNA library (generously provided by
R. Barstead and R. Waterston). The 59 ends of the cDNA were cloned
by RT-PCR using primers to unc-115 and the trans-spliced leader
sequence SL1 (Krause and Hirsh, 1987). Three classes of SL1spliced unc-115 transcripts were identified. An additional unc-115
cDNA, yk97f4.5, identified by Y. Kohara, has an alternate 59 upstream
exon. A full-length unc-115 cDNA was assembled from these fragments.
GFP fusions were made using expression vectors generously provided by Andrew Fire (Carnegie Institute of Washington). Germline
transformation and integration of transgenic arrays were performed
as described by Mello and Fire (1995) with lin-15(1) DNA as a coinjection marker. GFP fusion genes were integrated before characterization. For the unc-115 minigenes, the osm-6 promoter (Collet et
al., 1998) and the attenuated unc-115 promoter were isolated by
PCR. At least three independent lines expressing each transgene
were characterized.
Acknowledgements
We thank Shannon Grantner and Liqin Tong for excellent technical
assistance, Andrew Fire, Noelle L’Etoile, and Joe Chou for providing
unpublished clones, Jen Zallen for helpful observations, Tim Yu for
confocal assistance, and Sue Kirch, Erin Peckol and Jen Zallen for
comments on the manuscript. Some nematode strains used in this
work were provided by the Caenorhabditis elegans Genetics Center. The work was supported by the Howard Hughes Medical Institute and by National Institutes of Health grants GM22387 (to R. K. H.)
and HD22163 (to J. E. S.). E. A. L. was supported by the Howard
Hughes Medical Institute and a Damon Runyon–Walter Winchell
Cancer Research Fund fellowship. C. I. B. is an Assistant Investigator
of the Howard Hughes Medical Institute.
Received April 13, 1998; revised June 1, 1998.
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GenBank Accession Number
The accession number for the unc-115 cDNA sequence reported in
this paper is AF068867.