1 Department of Molecular, Cellular and Developmental Biology, Sinsheimer
Laboratories, University of California, Santa Cruz, CA 95064, USA
2 Department of Genetics, Yale University School of Medicine, New Haven, CT
06520-8005, USA
3 Howard Hughes Medical Institute, University of California, Santa Cruz, CA
95064, USA
* Present address: Fox Chase Cancer Center, Medical Oncology, 7701 Burholme
Avenue, Philadelphia, PA 19111, USA
Author for corrrespondence (e-mail:
jin{at}biology.ucsc.edu)
Accepted 31 March 2003
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SUMMARY |
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Key words: ADAM, Axon guidance, Migration, C. elegans, UNC-71, ADM-1, Netrin, Integrin
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INTRODUCTION |
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Most biochemical studies have focused on two functional modules of ADAM
proteins: proteolysis of membrane receptors by the metalloprotease domain, and
interaction with integrins by the disintegrin domain. The metalloprotease
domain is composed of 200 amino acids, and protease activity depends on a
conserved zinc-binding catalytic site sequence HExGHxxGxxHD, in which the
three histidines bind zinc and the glutamic acid is the catalytic residue.
Proteolysis of extracellular domains of transmembrane proteins by ADAMs, a
process also known as ectodomain shedding, has been shown to be important for
several signaling events (Black and White,
1998
). For example, the Drosophila Kuzbanian and its
orthologs in other species (Kuz/ADAM10 family) process the Notch receptor and
its ligand Delta in a conserved manner
(Rooke et al., 1996
;
Pan and Rubin, 1997
;
Qi et al., 1999
;
Lieber et al., 2002
). Kuz also
modulates axon repulsion by releasing the ephrin ligand
(Hattori et al., 2000
), and
axon extension, possibly through shedding and releasing of the netrin receptor
DCC (Fambrough et al., 1996
;
Galko and Tessier-Lavigne,
2000
). However, nearly half of the known ADAMs have amino acid
substitutions within the catalytic site sequence, and are likely to be
inactive metalloproteases (Black and White,
1998
). It has been proposed that inactive ADAMs may regulate the
activity of an ADAM with active protease through protein complex formation
(Pan and Rubin, 1997
). For
example, fertilin ß/ADAM2, an inactive metalloprotease, forms a
heterodimeric protein complex with fertilin
/ADAM1, an active ADAM, and
functions in sperm-egg fusion (Waters and
White, 1997
; Cho et al.,
2000
). However, definitive evidence for this hypothesis is still
lacking.
The disintegrin domain, which is named after the soluble snake venom (SVMP)
disintegrin (Gould et al.,
1990), is composed of about 100 amino acids, within which is a
disintegrin loop containing three invariant cysteines
(CX5-6CX5C)
(Wolfsberg et al., 1995
). In a
canonical disintegrin domain in SVMPs, the disintegrin loop has the
integrin-binding RGD tripeptide and can inhibit integrin functions in vitro.
However, only one ADAM has the RGD tripeptide, and the others have divergent
sequences that usually contain a conserved Arg next to the first cysteine and
a conserved aspartic acid next to the second cysteine. The interaction between
non-RGD containing ADAMs with integrins is not well understood. It has been
shown that fertilin ß, ADAM9, ADAM12, ADAM23 and ADAM28 can bind many
integrins (reviewed by Evans,
2001
), and that integrin
9ß1 can bind all ADAMs except
ADAM10 and ADAM17 (Eto et al.,
2002
). The functions of the cysteine-rich region and other domains
of ADAMs are much less studied, although it has been suggested that the
cysteine-rich domain may mediate cell fusion and adhesion, and that the
prodomain may regulate the protease activity
(Primakoff and Myles,
2000
).
The nematode C. elegans has four ADAM genes (adm-1, adm-2,
adm-4 and sup-17), and one secreted ADAM-like gene
(mig-17) (The C. elegans
Genome Consortium, 1998). SUP-17 is the Kuz/ADAM10 ortholog and
regulates LIN-12/Notch signaling pathway in vulva formation
(Wen et al., 1997
). MIG-17 is
required for the distal tip cell migration during gonadogenesis
(Nishiwaki et al., 2000
). We
report that the unc-71 gene corresponds to adm-1, which has
previously been identified through homology search
(Podbilewicz, 1996
).
unc-71 was defined by a single allele in the original general screen
for C. elegans mutants (Brenner,
1974
). Since then, a number of different screens have identified
many additional alleles of unc-71
(Chen et al., 1997
;
Huang et al., 2002
). Previous
characterizations of unc-71 mutants have revealed important functions
of unc-71 in various aspects of axon guidance, including axon
fasciculation of the ventral and dorsal nerve cords, and axonal morphogenesis
of the HSNs and the phasmid neurons
(Siddiqui, 1990
;
Siddiqui and Culotti, 1991
;
McIntire et al., 1992
).
unc-71 also plays a role in one of the mechanisms known to help guide
the migrations of the hermaphrodite sex myoblasts
(Chen et al., 1997
). The
unification of the genetic and phenotypic characterizations of unc-71
alleles with the molecular identity of unc-71/adm-1 has allowed us to
undertake a more detailed analysis of the role of this ADAM in these important
processes.
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MATERIALS AND METHODS |
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Molecular biology of unc-71
Cosmids and YAC clones were obtained from the Sanger Centre (Hinxton, UK).
DNA manipulations were performed following standard procedures
(Sambrook et al., 1989). The
unc-71/adm-1 genomic region was amplified using long range PCR kit
(Roche, Indianapolis, IN). The DNA pool that rescued unc-71 contained
three overlapping PCR products that covered the entire coding region with 6.6
kb 5' upstream and 0.7 kb 3' downstream sequences. unc-71
minigene (pCZ418) was generated by placing into the cDNA yk344a1 the
EcoRV genomic fragment (47 to +1641) containing the first
intron and the KpnI-SpeI genomic fragment (+3132 to +7149)
containing the last intron, last exon and 1018 bp 3' UTR. To generate
the unc-71 metalloprotease domain deletion clone (pCZ423), a 441 bp
BstZ17I-SspI fragment in pCZ418 was deleted. To generate the
unc-71 C-terminal truncation clone, a 3.3 kb
BbsI-SpeI fragment in pCZ418 was deleted. A second
unc-71 minigene (NH#1092) was constructed by cloning the
BglII-MfeI (132 to +1795) genomic fragment and the
MfeI(+236)-ApaI (a site in the vector) cDNA fragment from
yk344a1 clone into the BamHI-ApaI sites of pBluescript II
KS(+). The two minigenes behaved very similarly in transformation rescue
assays. To generate the unc-71 transcriptional GFP construct pCZ411,
the 1.7 kb upstream regulatory region of unc-71 was amplified by PCR,
and inserted into pPD95.79. For UNC-71::GFP fusion, the GFP-coding sequence
from pPD114.35 was inserted at the BclI site in pCZ418, which
resulted in in-frame fusion of GFP in the cytoplasmic region of UNC-71.
Germline transformation was performed following standard procedures
(Mello et al., 1991) using
10-20 ng/µl of unc-71 DNAs and 50-100 ng/µl pRF4 co-injection
marker. The Punc-71GFP extrachromosomal arrays were obtained by
co-injecting pCZ411 along with the 6.6 kb promoter generated by PCR. One
integrant (juIs166) of Punc-71GFP extrachromosomal arrays
was obtained by a Psoralen-UV induced mutagenesis, and backcrossed multiple
times.
To identify lesions in unc-71 alleles, unc-71 genomic DNA, including all exons and exon-intron junctions were amplified from unc-71 mutant and wild-type animals. DNA sequences were determined using 33P labeled primers and the fmol sequencing kit (Promega, Madison, WI). All lesions were confirmed on both strands and from DNAs prepared in independent PCRs.
To verify the effect of ju156 mutation on unc-71 mRNA, RT-PCR was performed on total RNA isolated from unc-71(ju156) animals. Two transcripts were found, one is as predicted by the deletion; the other uses a new splice donor before the deletion. In both transcripts, the reading frame is altered and translation would terminate prior to the metalloprotease domain.
Tissue-specific expression of unc-71
The Punc-115, Pjam-1, Punc-119,
Punc-33, PF25B3.3, Punc-25, Punc-30 and
Pglr-1 promoters (Jin et al.,
1994; Hart et al.,
1995
; Maduro and Pilgrim,
1995
; Lundquist et al.,
1998
; Jin et al.,
1999
; Altun-Gultekin et al.,
2001
; Koppen et al.,
2001
) were inserted upstream of the unc-71 mini-gene in
pCZ418. Other tissue-specific promoters were obtained by PCR amplification to
yield fragments that span the following genomic regulatory regions (the
numbers indicate the basepair relative to the ATG): Ptwist, from
1334 to 13; Psur-5, from 3711 to 1;
Pmyo-3, from 2383 to 1
(Okkema et al., 1993
;
Harfe et al., 1998
;
Yochem et al., 1998
). The
Pe15*2 promoter contains a 234 bp fragment from the
egl-15 promoter region (from 1530 to 1296) that was
inserted upstream of the minimal pes-10 promoter of pPD97.78 (a gift
from A. Fire) between the HindIII and StuI sites.
Tissue-specific promoters were inserted into the multiple cloning sites
upstream of the unc-71 minigene in NH#1092.
Pe15*2unc-71
was generated by cloning the 5 kb SpeI/ApaI unc-71
minigene from NH#1092 into the Pe15*2-containing construct
NH#1134.
Transformation rescue of the SM defects
The sem-5(n1779) mutant background was used to reveal the effects
of unc-71 mutations on SM migration. In this background, compromised
unc-71 function causes the animal to have a high-penetrance Egl
phenotype and posteriorly displaced SMs. It also significantly enhances the
Unc phenotype (Chen et al.,
1997). YAC germline transformation rescue was assayed based on the
Unc and Egl phenotypes of unc-71(ay7); sem-5(n1779). Transgenic lines
were obtained using the co-transformation marker rol-6(su1006) in
plasmid pRF4. unc-71 animals attempt to, but cannot complete, the
rolling motion. Lines were first scored for rescue of the Unc phenotype;
nonUnc lines were then scored for the penetrance of their Egl phenotype.
unc-71 minigenes and constructs where unc-71 was expressed
from tissue-specific promoters were assayed directly for rescue of the
posterior SM positioning defect of unc-71(ju156); sem-5(n1779)
animals. The failure of rescuing the Egl phenotype of this strain by these
constructs may be due to multiple egg-laying defects in several cell types
that are not rescued by tissue-specific expression of unc-71 or
non-optimal expression of unc-71 from these transgenic arrays. Most
constructs were injected into unc-71(ay7); sem-5(n1779) at 50
µg/ml along with pJKL449.1 [Pmyo-2GFP] at 5 ng/µl; some were
injected into unc-71(ay7); dpy-20(e1282ts); sem-5(n1779) with pMH86
[dpy-20(+)] at 50 ng/µl. The penetrance of the egg-laying defect
(%Egl) is scored as the percentage of Egl animals 48 hours after the L4 stage
(n>30). SMs are scored with respect to the nuclei of the ventral
hypodermal Pn.p cells as described (Thomas
et al., 1990
).
GFP and motoneuron phenotype analysis
All GFP markers and unc-71 GFP transgenes were directly observed
under a 63x objective on a Zeiss Axioskop fluorescence microscope
equipped with a HQ-FITC filter (Chroma, Brattleboro, VT). The type D
motoneuron defects were scored using juIs76 marker as follows. In
wild-type L1 animal, the commissures of all six DDs reach the dorsal cord, and
DD1 commissure runs on the left side while the other five on the
right side. This pattern was scored as 0% commissure outgrowth defect, 0%
circumferential guidance defect and 16.7% (1/6) of LR error. In
unc-71 mutant L1 animals, the commissures that failed to exit the
ventral cord were scored as commissure outgrowth defects; the commissures that
grew out, but did not reach the dorsal cord, were scored as circumferential
guidance defects; and the commissures that run on the opposite sides compared
with wild type were scored as LR error. In L2 to adult animals, the same
categories were scored for DD2-6 and VD1,3-13. 0% of commissures
(0/17) run on the left side in wild-type animals.
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RESULTS |
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unc-71 was previously mapped to the right arm of chromosome III
between dpy-18 and unc-25, and is covered by deficiency
tDf6, but not ctDf3
(Stein et al., 2001). We
further located unc-71 between SNPs(III) +17.4 and
+20.1 using the snip-SNP mapping strategy
(Wicks et al., 2001
) (see
Materials and Methods). YAC clones in this region were tested for germline
transformation rescue of the severe Unc and egg-laying defective (Egl)
phenotypes owing to the unc-71(ay7) mutation in a sensitized
sem-5(n1779) mutant background
(Chen et al., 1997
). Two
overlapping YACs, Y37D8 and Y52B8, were found to rescue both phenotypes of
unc-71(ay7); sem-5(n1779) (Fig.
1A).
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unc-71 mutations predominantly affect the disintegrin and
cysteine-rich domains
ADM-1 was originally identified from a homology search using conserved
sequences of the ADAM family of proteins
(Podbilewicz, 1996). It has
all the signature domains of ADAM proteins with a relatively long cytoplasmic
tail. The metalloprotease domain of ADM-1 is likely to be inactive as it has a
glutamine in the first zinc-binding histidine position and a serine in the
catalytic residue glutamic acid position
(Fig. 1B). The disintegrin loop
of ADM-1 has a PCD tripeptide at the position that may correlate to the RGD
tripeptide in SVMP (Fig. 1B).
Overall, UNC-71/ADM-1 is more closely related to the mind-meld gene
of Drosophila than to other ADAMs, but defines a unique member called
ADAM14
(www.gene.ucl.ac.uk/nomenclature/genefamily/metallo.html).
Throughout the paper, we refer to this gene as unc-71.
To investigate the functional requirement of different domains in the UNC-71 protein, we first determined the molecular lesions in unc-71 alleles. ju156 is a 170 bp deletion within the second exon that results in a frameshift followed by a premature stop before the metalloprotease domain (Table 1). Using RT-PCR on RNA isolated from unc-71(ju156) mutants, we did not detect any transcripts that would produce functional proteins. Therefore, ju156 is most probably a null allele of unc-71. ju161 is a missense mutation changing a Gly to an Arg in the prodomain. Most of the lesions in other unc-71 alleles are clustered in the disintegrin and the cysteine-rich domains (Table 1), providing strong evidence that these two domains are required for UNC-71 function in vivo. More than half of the mutations are missense mutations that might affect specific interactions with binding partners, or protein conformation and stability. Among the six mutations in the disintegrin domain, ju159 and ju160 affect two conserved residues in the disintegrin loop, changing Cys509 to Tyr509 and Asp504 to Asn504, respectively (Fig. 1B), supporting a pivotal role of this loop in ADAM proteins. e541, ju157 and ay47 are three missense mutations in the cysteine-rich domain. ay44 alters a conserved Cys in the EGF repeat. ju255, a weak allele, and ay48, a strong allele, both have lesions in the cytoplasmic domain, with ju255 changing Arg990 to Lys990 and ay48 generating a stop codon that would delete the last 94 amino acids.
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The unc-71 promoter is active in a selected set of cells
including the excretory cell and glands, some neuronal and epidermal
cells
Antibodies raised against the cytoplasmic domain of ADM-1 detected protein
expression in multiple tissues, including epidermis, pharynx, vulva and sperm
(Podbilewicz, 1996). However,
this expression pattern is largely unaltered in unc-71(e541) and
unc-71(ju156) animals, suggesting that the antibodies may recognize
an unrelated protein(s) in addition to UNC-71 (B. Podbilewicz, personal
communication).
To determine the cell types in which unc-71 is expressed, we
expressed GFP (Chalfie et al.,
1994) under the control of the unc-71 promoter using the
combination of the 1.7 kb short promoter along with the 6.6 kb long promoter
(see above, and Materials and Methods). GFP expression was first observed in
several posterior cells of comma stage embryos, then in the excretory cell and
some head neurons in threefold stage embryos (data not shown). In L1 to adult
worms, continued expression of GFP was observed in several head neurons, the
excretory cell and the excretory gland cells, as well as the sphincter muscles
(Fig. 2A-E). We identified one
of the head neurons as the AVG neuron based on the cell position and
morphology (Fig. 2C). In
addition, in L4 worms, GFP was seen in a set of hypodermal cells surrounding
the vulva, which persisted in the hypodermal cells flanking the vulval opening
in adult worms (Fig. 2E). No
GFP expression was detected in the sensory neurons. Faint GFP expression was
infrequently detected in one to four neurons in the ventral cord. No
expression was observed in the sex myoblasts, nor was GFP detected in the body
wall muscles at any stages.
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unc-71 acts cell non-autonomously for sex myoblast
migration
The restricted expression pattern of Punc-71GFP raises the
question where its function is required for sex myoblast migration and motor
axon guidance. We first addressed this question by using a variety of
tissue-specific promoters to drive expression of the unc-71 mini-gene
and assaying its function in sex myoblast migration.
unc-71 plays an important role in one of the mechanisms used to
guide the migrations of a pair of hermaphrodite sex myoblasts (SMs), the
progenitors of the egg-laying muscles
(Chen et al., 1997;
Chen and Stern, 1998
). The SMs
migrate anteriorly from the posterior body region to positions that flank the
precise center of the gonad (Sulston and
Horvitz, 1977
). The gonad attracts the SMs to their precise final
positions by means of a fibroblast growth factor chemoattractant, a mechanism
termed the gonad-dependent attraction (GDA)
(Burdine et al., 1998
). In the
absence of the gonad, the SMs still migrate anteriorly and end up within a
range of positions that spans the center of the animal. The mechanism
responsible for this movement is known as the gonad-independent mechanism
(GIM). Mutations in unc-71, as well as in two other genes,
unc-73 and unc-53, compromise the GIM
(Chen et al., 1997
). As long
as the gonadal chemoattraction remains, mutations in these genes do not
significantly alter the final positions of the SMs. However, when the GDA is
compromised, mutations in these genes dramatically affect the ability of the
SMs to migrate anteriorly and cause the SMs to remain in severely posteriorly
displaced positions. Therefore, to assess the ability of unc-71 to
function in the GIM, a sem-5 mutant background was used to eliminate
the masking effects of the GDA chemoattractive mechanism
(Chen et al., 1997
).
Although the two YACs containing unc-71 rescued both the Egl and Unc phenotypes of the unc-71; sem-5 double mutant, no smaller constructs were found to rescue the Egl phenotype efficiently (Fig. 1A). Therefore, we determined the rescue of the SM migration defect by directly analyzing the distribution of the final positions of SMs in transgenic lines (see Materials and Methods). SM distributions that resembled that of the sem-5 single mutant were classified as fully rescued, while transgenic lines with distributions resembling the unc-71; sem-5 double mutant were scored as failing to rescue. Intermediate distributions were classified as showing partial rescue. Examples of these classes of rescue are shown in Table 3A (Psur-5, full rescue; Punc-119, partial rescue; Pmyo-3, no rescue).
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Further examination of axon guidance of DD and VD motoneurons in
unc-71 mutants
Several neuronal defects in unc-71 mutants have been reported
previously (Siddiqui, 1990;
Siddiqui and Culotti, 1991
;
McIntire et al., 1992
). To
extend these studies, we examined the expression patterns of a panel of GFP
markers that visualize either the entire nervous system (Punc-119GFP)
or specifically in subsets of neurons, including the touch neurons
(Pmec-7GFP), the interneurons (Pglr-1GFP), the AWC sensory
neurons (Pstr-2GFP), the ventral cord cholinergic motoneurons
(Pacr-2GFP) and the GABAergic motoneurons
(Punc-25GFP). We observed comparable defects in animals homozygous
for ju156, ay7 or e541. Overall, longitudinal nerve bundles
are mildly defasciculated (Fig.
3I,J), many neurons exhibit low penetrance of premature axon stop
and branching errors (10-20% of axons for a given type of neurons,
n>50 mutant animals for each GFP marker). For example, 18% of the
ASI sensory neurons showed premature stop, and 4% showed axon wander
(Fig. 3L). Some neuron cell
bodies are occasionally mispositioned (data not shown). A striking
differential effect of unc-71 was observed on the different classes
of the ventral cord motoneurons. The cholinergic motoneurons showed less than
10% axon guidance errors (Table
2; see Fig. 5L),
whereas the GABAergic type D motoneurons showed fully penetrant defects in
several aspects of axon guidance, as described in detail below.
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We categorized the axon defects of the type D neurons in unc-71 mutants into following classes (Table 2, also see Materials and Methods): (1) defasciculation of longitudinal axons (Fig. 3F); (2) failure of commissural outgrowth (Fig. 3D); (3) error in the sidedness of commissures (Fig. 3H); (4) premature stall and wander of commissures (Fig. 3B,H); and (5) abnormal trajectory of the VD13 neuron (Fig. 3K). We collected most of the quantitated data from unc-71(ju156) and unc-71(ju255) animals, and also made qualitative observations in other strong loss-of-function unc-71 mutants. In L1 larvae of unc-71(ju156), the ventral longitudinal processes of DD neurons grew to their normal lengths; however, 33% of the DD neuron commissures did not exit the ventral cord (scoring six commissures/animal; n=31) (Fig. 3C,D). Of the remaining commissures (average four/animal), 32% run on the left side of the body (range from 0-3, average 1.3), and 21% stopped prematurely before reaching the dorsal cord and wandered in the lateral regions of the body. In L2 to adult mutant animals, the ventral longitudinal nerve bundles of DD and VD neurons were defasciculated (36.4%, scoring 10 VD neurons/animal, n=14) (Fig. 3E,F). Thirty-nine percent of all commissures failed to exit the ventral cord (scoring 17 commissures/animal, n=43). Of the remaining ones (average 10.6 commissures/animal), 39% run on the left side of the body (range from 1-9, average 4.1), and 21% stopped prematurely and wandered. The failure in commissural growth also left axonal gaps in the dorsal cord; on average, 2.6 axon gaps were seen at various positions in the dorsal cord (Fig. 3G,H). The comparison of the defects in adults with those for DD neurons in L1 larvae indicates that the VD neurons are affected equally as the DDs in unc-71 mutants. Last, VD13, the most posterior VD neuron, showed distinct defects in that instead of sending a process anteriorly, it always sent out a process posteriorly, which either stalled and extended numerous branches in the tail (38%, n=63), or grew dorsally to join the dorsal cord where it looped back extending anteriorly and forming a left-side-down U shape (62%) (Fig. 3K).
In the weak allele unc-71(ju255) animals, the major defects were seen in VD13 trajectory and in the sidedness choice for commissures (Table 2). Ninety-four percent of the commissures exited the ventral cord, 30% of which run on the left side, and only 4% did not reach the dorsal cord (n=30). The phenotypes in ju255 suggest that the decision of the growth cones at the commissural exit choice is more sensitive to unc-71 activity than those for longitudinal axon fasciculation and circumferential growth.
unc-71 acts cell non-autonomously to regulate D motor neuron
axon guidance
To address where UNC-71 function is required for D neuron axon guidance, we
first performed genetic mosaic analysis with unc-71(ju156);
juIs76[Punc-25GFP] animals that also carried an extrachromosomal array
containing the unc-71 rescuing DNA pool and SUR-5::GFP, a
cell-autonomous nuclear GFP marker (Yochem
et al., 1998). The AB lineage generates neurons and hypodermis,
and the P1 lineage produces all but one muscle
(Sulston et al., 1983
). We
found eight mosaic animals that lost the unc-71(+) array in the AB,
but not the P1, lineage, and all eight animals exhibited axon defects in the D
neurons. By contrast, 10 animals that lost the array in the P1, but not the
AB, lineage showed wild-type axon morphology of the D neurons. This analysis
indicates that unc-71 function is required in the neuro-hypodermal
lineage for motor axon guidance. Consistently, when unc-71 was
expressed from the unc-115 promoter
(Lundquist et al., 1998
), it
fully rescued the D neuron axon phenotypes
(Table 4).
In which cells of AB lineage does unc-71 act? We expressed
unc-71 pan-neurally using three promoters: Punc-119
(Maduro and Pilgrim, 1995),
Punc-33 and PF25B3.3
(Altun-Gultekin et al., 2001
)
and observed a partial rescue of the D neuron axon defects
(Table 4A). We also expressed
unc-71 in all epidermal cells using the Pe15*2
promoter, and observed similar rescuing activity. By contrast, expression of
unc-71 from the myo-3 muscle promoter
(Okkema et al., 1993
) did not
show any rescuing activity. These data further support that unc-71
function is required in both neurons and epidermis for motor axon
guidance.
In which neurons does unc-71 act? We expressed unc-71
specifically in the D neurons using the unc-30 or unc-25
promoter (Jin et al., 1994;
Jin et al., 1999
), and found
that neither transgenes rescued the axon defects
(Table 4), indicating that
unc-71 may act in other neurons to control D neuron axon guidance.
Expression of unc-71 in cholinergic ventral cord motoneurons using
the acr-2 promoter did not rescue the D neuron axon defects. By
contrast, expression of unc-71 from the glr-1 promoter,
which is active in several interneurons including the AVG neuron
(Hart et al., 1995
), partially
rescued the axon defects of the D neurons in unc-71 mutants to the
same extent as Punc-119 UNC-71
(Table 4A). unc-71 is
expressed in AVG (Fig. 2C). We
do not rule out that the expression of unc-71 in other
glr-1-expressing neurons contributes to guide the D neuron axons.
Nonetheless, these data support the conclusion that unc-71 functions
cell non-autonomously for motoneuron axon guidance, and suggest that one
possible neuronal source of UNC-71 is from the AVG neuron.
Genetic interactions of UNC-71 with other ADAMs indicate that UNC-71
does not act through other active ADAMs
It has been suggested that an ADAM protein with an inactive metalloprotease
domain could function as an endogenous inhibitor to regulate the activity of
an ADAM with an active metalloprotease
(Pan and Rubin, 1997). The
C. elegans genome has four ADAMs
(The C. elegans Genome
Consortium, 1998
). Except for UNC-71, the other three ADAMs
(ADM-2, SUP-17 and ADM-4) all contain active catalytic site sequences in their
metalloprotease domains (Wen et al.,
1997
) (B. Podbilewicz, personal communication; this study). If
unc-71 acts as an inhibitor for these active ADAMs, we would expect
that some of the phenotypes in unc-71 might be due to hyperactive
metalloprotease activities of the other ADAMs, and therefore, could be
suppressed by eliminating or reducing the function of these active ADAMs.
We first examined the D neuron morphology in the other ADAM mutants. Animals homozygous for adm-2(tm347) or adm-4(ok265), both of which are deletion mutations (see Materials and Methods), are viable, fertile, have no obvious behavioral defects, and show no defects in D neuron morphology (Fig. 4A). sup-17(n1258ts) is a temperature-sensitive mutation, and the animals are viable at 15-22.5°C, but lethal at 25°C. In sup-17(n1258ts) animals cultured at 22.5°C, we observed a very weak D neuron axon guidance defect such that about 4% of the commissures failed to reach the dorsal cord (Fig. 4A).
|
unc-71 acts in parallel to ina-1/pat-3 integrins in
motor axon guidance
It is generally thought that a major function of ADAM proteins is to
regulate the extracellular matrix, and that the integrin receptors are
important targets of ADAM proteins
(Wolfsberg et al., 1995;
Evans, 2001
). The C.
elegans genome has two integrin-
subunits, ina-1 and
pat-2; one integrin-ß subunit, pat-3; and one
integrin-ß subunit, inb-1
(Hutter et al., 2000
). INA-1
can form a protein complex with PAT-3, and is expressed and functions
cell-autonomously in neurons and migrating cells
(Baum and Garriga, 1997
). The
ina-1-null mutants are L1 lethal, and exhibit weak defasciculation
and commissural outgrowth defects in the D neurons
(Baum and Garriga, 1997
)
(Fig. 4B). pat-2 and
pat-3 are reported to be expressed predominantly in muscles and
gonad, and may form a complex (Williams
and Waterston, 1994
; Gettner
et al., 1995
). Null mutants of pat-2 or pat-3
are arrested as paralyzed twofold stage embryos
(Williams and Waterston,
1994
), precluding us from determining the neuronal phenotypes in
these animals. In partial loss-of-function mutation
pat-3(rh151) animals, we observed that the D neurons
displayed a low level of defasciculation and commissure outgrowth defects
(Fig. 4B). Similar defects have
also been reported by a study using pat-3 RNAi
(Poinat et al., 2002
). INB-1
is a rather divergent member of integrins. The inb-1(tm353) mutants,
a likely knockout mutation (see Materials and Methods), are viable, fertile,
have no obvious behavior defects and their D neuron morphology is normal
(Fig. 4B).
To investigate how unc-71 interacts with the C. elegans
integrins, we made double mutants of unc-71 with viable alleles of
these integrins. inb-1(tm353); unc-71(ju156) double mutants showed
similar axon defects as unc-71(ju156) alone
(Fig. 4B), suggesting that
inb-1 acts in a separate process from unc-71. By contrast,
unc-71 showed synergistic interactions with ina-1 and
pat-3 in longitudinal axon fasciculation. For example, in adult
animals of ina-1(gm144), 15% of the longitudinal processes of D
neurons showed defasciculation (scoring 10 VD neurons/animal, n=15),
about 16% of the commissures run on the left side, but most commissures
reached the dorsal cord (98%, 17 commissures/animal, n=27)
(Fig. 4B)
(Baum and Garriga, 1997).
Although gm144 is a partial loss-of-function mutation of
ina-1 (Baum and Garriga,
1997
), the axon defects of the DD neurons in L1 larvae of
ina-1(gm144) were comparable with those of gm86 (data not
shown), a null allele of ina-1, suggesting that the D neuron
phenotypes we scored here reflect the null phenotypes of ina-1 in
these neurons. In the unc-71(ju156); ina-1(gm144) double mutants, the
ventral fascicle of the D neurons were severely disorganized
(Fig. 5J): 90% of the
longitudinal D axon bundles were defasciculated (10 VD neurons/animal,
n=18), compared with 37% and 15% for unc-71(ju156) and
ina-1(gm144) alone. Likewise, 80% of the D neurons showed
defasciculation in unc-71(ju156); pat-3(rh151) animals, compared with
37% and 6% for unc-71(ju156) and pat-3(rh151) alone. In
addition, greater number of commissures failed to reach the dorsal cord in
these double mutants than each single mutant
(Fig. 5D). The phenotypic
similarities in unc-71; ina-1 and unc-71; pat-3 double
mutants lend further support for the hypothesis that INA-1 and PAT-3 are
functional partners in vivo. Moreover, the enhanced defects in the double
mutants of unc-71 with ina-1 or pat-3 suggest that
unc-71 is unlikely to act through ina-1/pat-3 integrins;
rather, it acts redundantly, or in parallel to, ina-1/pat-3 to
control axon fasciculation.
unc-71 likely acts in parallel to netrin-induced axon
repulsion
It has been shown that chemical inhibitors of metalloproteases can
potentiate netrin-mediated axon outgrowth and guidance, and the netrin
receptor DCC appears to be a substrate for metalloprotease-dependent
ectodomain shedding (Galko and
Tessier-Lavigne, 2000). Drosophila kuz mutants exhibit
axon extension defects, the mechanism for which is not known
(Fambrough et al., 1996
). The
circumferential guidance of D neuron commissures is repelled by the
UNC-6/netrin. UNC-6 acts upon the UNC-5 receptor and the UNC-5 and UNC-40/DCC
co-receptor (Wadsworth, 2002
).
The signal transduction pathway downstream of the receptors is in part
mediated by unc-34/Ena and max-1
(Colavita and Culotti, 1998
;
Huang et al., 2002
). In
mutants for each of these genes, the circumferential axon guidance of D
neurons is disrupted to different extents
(Hedgecock et al., 1990
;
McIntire et al., 1992
;
Colavita and Culotti, 1998
;
Huang et al., 2002
). To
explore potential interactions between unc-71 with
unc-6/netrin signaling pathway, we conducted double mutant analysis
as below.
In null mutants for unc-6 or unc-5, about 35% or 26% of
the D neuron commissures grew out of the ventral cord, respectively; but none
reached the dorsal nerve cord. In unc-71(ju156) animals, 61% of the D
neuron commissures grew out of the ventral cord, of which 21% failed to reach
the dorsal nerve cord. In both unc-71(ju156); unc-5(e53) and
unc-71(ju156); unc-6(ev400) double mutants, no commissures of the D
neurons grew out of the ventral cord (Fig.
5F). Moreover, this complete block of commissural outgrowth in the
double mutants is specific to the type D neurons because the commissural
outgrowth and guidance of the DA and DB motoneurons, which also depends on
unc-6 and unc-5
(Hedgecock et al., 1990), was
comparable between the double mutant animals and unc-5 or
unc-6 single mutants alone (Fig.
5M,N). These observations indicate that unc-71 and
unc-6 act in parallel to control commissural outgrowth.
To examine how unc-71 interacts with the unc-6/netrin signaling pathway in circumferential axon guidance, we used partial loss-of-function mutations of unc-5 and unc-6. In these animals, all D neuron commissures exit the ventral nerve cord, and only a fraction of them exhibited circumferential guidance defects; for example, 21% and 15% for unc-5(ju181) and unc-6(ju152), respectively (Fig. 4C). In unc-71(e541); unc-5(ju181) and unc-71(e541); unc-6(ju152) double mutants, however, the circumferential guidance defects were strongly enhanced to 87% and 90%, respectively (Fig. 4C). Similar enhancement was also observed for double mutants between unc-71 with unc-40, or unc-34, or max-1 (Fig. 4C). We also noticed that the final positions of the stalled growth cones in the double mutants resembled those in the netrin signaling mutants more than those in unc-71 mutants. For example, in unc-71(ju156 or e541) single mutants, most stalled growth cones were found within the dorsal half along the side of the worm, whereas in unc-5(ju181) and unc-5(ju181); unc-71(ju156) double mutants, the growth cones were stalled at various positions along the dorsoventral axis. Similarly, the stalled growth cones were often found at mid-lateral range along the dorsoventral axis for unc-40(e1430) and unc-40(e1430); unc-71(ju156) double mutants, and at ventral half in max-1(ju39) and max-1(ju39); unc-71(ju156) double mutants. Because the double mutants between unc-71 with netrin signaling molecules all showed an overall additivity to enhancement, we interpret these results to mean that unc-71 functions in parallel to netrin-mediated commissural outgrowth and circumferential guidance. However, the observation that the final positions of the stalled growth cones in the double mutants resemble those of netrin signaling pathway mutants alone may imply that unc-71 modulates the cellular motility of growth cones through events downstream of netrin receptors.
![]() |
DISCUSSION |
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A crucial function of UNC-71 is in cell adhesion
The ADAM proteins are composed of multiple protein functional modules.
Current studies of ADAMs have focused on the metalloprotease and the
disintegrin domains. Our analysis of the many alleles of unc-71 and
transgene studies provides a comprehensive in vivo examination of the
structural and functional correlation of an ADAM protein.
On the basis of the sequence comparison, UNC-71 is unlikely to be an active
metalloprotease (Fig. 1A)
(Podbilewicz, 1996). We find
that none of the large number of existing unc-71 mutations has
missense mutations in this domain. However, deletion of this domain abolishes
UNC-71 rescuing activity. It has been shown that the metalloprotease domain of
MIG-17 is required for MIG-17 localization
(Nishiwaki et al., 2000
). It
is possible that the inactive protease domain of UNC-71 may be necessary for
maintaining the structural integrity, or for facilitating post-translational
processing for proper targeting of the protein.
An important finding of our mutation analysis is that the majority of
unc-71 mutations are missense mutations altering conserved residues
in the disintegrin and cysteine-rich domains, lending strong support to the
hypothesis that these domains are important functional modules. In particular,
in vitro studies have indicated the importance of an aspartic acid residue in
the disintegrin loop of ADAMs for their interactions with integrins
(Zhu et al., 2000). We find
that the ju160 mutation alters this Asp residue, and behaves as a
null mutation. The observation that expression of the extracellular region of
UNC-71 in wild-type animals weakly phenocopies the axon guidance defects seen
with partial loss-of-function mutations of unc-71 is also consistent
with the notion that UNC-71 is involved in a regulated cellular adhesion
process. Based on the lesions in unc-71 loss-of-function mutations,
we believe that a crucial role of UNC-71 is in cell adhesion.
unc-71 acts cell non-autonomously in D neuron axon guidance
and sex myoblast migration
Although UNC-71 appears to be expressed as a membrane protein and the
membrane anchorage is necessary to control its activity, we have substantial
evidence to support the conclusion that unc-71 functions in a cell
non-autonomous manner for motoneuron axon guidance and SM migration. First,
unc-71 shows restricted expression in a selected set of cells that do
not appear to include the motoneurons and sex myoblasts. Second, expression of
UNC-71 in the D neurons alone does not rescue the axon guidance defects; nor
does expression of UNC-71 in muscles rescue the migration defects of sex
myoblast. Third, our tissue-specific expression and rescue study shows that
the motoneuron axon guidance and SM migration depend on the expression of
unc-71 in epidermis and neurons. A cell non-autonomous function is
not unusual for membrane-anchored proteins. For example, Kuz has both
cell-autonomous and cell-non-autonomous roles in bristle development
(Rooke et al., 1996). Ephrins
are known to function cell non-autonomously in neuron for epidermal cell
migration in C. elegans (George
et al., 1998
). In light of the cell non-autonomous requirement for
unc-71, it is somewhat surprising to find that unc-71
mutations cause profound defects in the type D neurons without significant
effects on the other types of the ventral cord motoneurons, despite that these
neurons are born within close proximity and migrate along nearly identical
paths. This observation suggests that UNC-71 does not alter the extracellular
matrix in a global manner; rather, UNC-71 interacts with a receptor(s), or
alters the distribution of a ligand(s) for a receptor(s) on the type D
neurons.
The molecular identification of UNC-71 as an ADAM metalloprotease and the
possibility that it acts in a cell non-autonomous manner in SM migration is an
important step forward in understanding the control of SM migration guidance
by the gonad-independent mechanism (GIM). Little is known about the source of
guidance information for this mechanism. Three components of this mechanism
have been identified: UNC-53, UNC-73 and UNC-71
(Chen et al., 1997). Double
mutant combinations between any of the three genes unc-71, unc-73 and
unc-53 have SM distributions that are essentially wild type and
similar to the single mutants (E. Chen and M.J.S., unpublished), consistent
with these components acting in a single pathway that mediates the GIM. Both
UNC-53 and UNC-73 encode proteins containing structural domains or activities
that can modify the intracellular cytoskeleton, and they have cell-autonomous
function in other processes (Steven et
al., 1998
; Stringham et al.,
2002
). A cell non-autonomous role for UNC-71 in SM migration
provides a link to the extracellular cues involved in the gonad-independent
mechanism.
UNC-71 provides dynamic and distinct guidance cues for D motor
axons
Precise axon trajectory is achieved in a stepping stone manner, and growth
cones can detect specific guidance information at specific choice points or
guideposts (Tessier-Lavigne and Goodman,
1996). The C. elegans type D neurons have at least four
guideposts during their axon trajectory: longitudinal fasciculation, branching
of circumferential processes, circumferential guidance and bifurcation in the
dorsal cord. Mutations in several genes have been shown to affect one guidance
aspect predominantly. For example, ina-1 regulates the longitudinal
fasciculation (Baum and Garriga,
1997
); the UNC-6/netrin and its receptors UNC-5 and UNC-40
regulate the dorsal-directed commissural growth
(Wadsworth, 2002
). In
unc-71 mutants, we find a range of axon guidance defects that are
consistent with an interpretation that unc-71 is required in three
steps, with a major function in longitudinal fasciculation and commissural
branching, and a minor role in dorsal-directed guidance.
Axon fasciculation requires cell-cell interactions and is generally thought
to be mediated by short-range or contact-mediated guidance mechanisms. A
prominent defect of unc-71 mutants is defasciculation of the DD and
VD longitudinal processes. ADAM proteins are implicated in promoting cellular
adhesion mostly through the integrin receptors
(Evans, 2001). Integrins
function as
ß heterodimer receptors
(Miranti and Brugge, 2002
). In
C. elegans, PAT-3/ß-integrin can form dimers with INA-1 or PAT-2
integrins (Baum and Garriga,
1997
; Gettner et al.,
1995
). We could not address the interaction of unc-71
with pat-2 for the early lethality of pat-2 null mutants and
for the lack of viable alleles. The fasciculation defects in ina-1
and pat-3 mutants are much weaker than those in unc-71, and
are strongly enhanced in ina-1; unc-71 and pat-3; unc-71
double mutants. These observations are consistent with a conclusion that
UNC-71 does not act through these integrins; rather, it acts independently of,
or in parallel to, ina-1/pat-3 integrins in axon fasciculation.
The second guidepost for the D neurons is the branch choice of the
circumferential processes. In wild-type animals, the D neurons extend side
branches near the anterior end of the ventral processes, and there is a
striking asymmetry in that 17 out of the 19 commissures exit to the right side
of the animal. In unc-71 mutants, 39% of the branches fail to exit
the ventral cord, and 39% of the remaining ones choose the left side. This
branching defect is an unlikely secondary consequence caused by the
defasciculation defects of the ventral processes, because
unc-71(ju255) specifically disrupts the branch sidedness decision
without affecting fasciculation. Moreover, mutations in unc-5 and
unc-40 cause defasciculation of the ventral processes, but do not
affect the asymmetrical preferences of the commissural branches
(Hedgecock et al., 1990;
McIntire et al., 1992
) (this
study). Thus, the activity of UNC-71 appears to be more crucial for the branch
choice than for longitudinal fasciculation. It has been shown that killing the
AVG neuron, which projects a long process to pioneer the ventral cord and
fasciculates with the extending D processes, causes the branches of the D
neurons to exit to the left side (R. Durbin, PhD Thesis, University of
Cambridge, 1987). We find that unc-71 is expressed in AVG and that
the axon defects of the D neurons can be partially rescued by expressing
UNC-71 from the glr-1 promoter. These data support the hypothesis
that UNC-71 expressed from the AVG neuron may facilitate a localized guidance
source at the D neuron branch points. AVG also expresses UNC-6
(Wadsworth et al., 1996
), and
unc-6 mutants show commissural outgrowth failure and LR sidedness
errors (Hedgecock et al.,
1990
) (this study). We find that in unc-71; unc-6 and
unc-71; unc-5 double null mutants the commissural outgrowth is
completely blocked, indicating that UNC-71 acts in parallel to UNC-6 and
UNC-5. ina-1 mutants show LR sidedness errors
(Baum and Garriga, 1997
);
however, the commissural outgrowth defects in unc-71; ina-1 double
mutants are the same as in unc-71 mutant alone. Conceivably, UNC-71
may create a microdomain at the commissural exit guidepost to facilitate the
combinatorial interactions between these molecules.
The third guidepost for the D neurons is the dorsal-directed migration of
the commissures. UNC-6 provides a long-range repulsive cue, and acts through
its receptors UNC-5 and UNC-40 (Wadsworth,
2002). Both the unc-71 mutant phenotypes and the double
mutant analysis of unc-71 with genes involved in this UNC-6 repulsion
pathway indicate that unc-71 modulates this axon guidance process.
Although ectodomain shedding of netrin receptors and other transmembrane
receptors by ADAMs have been implicated in growth cone movement
(Fambrough et al., 1996
;
Galko and Tessier-Lavigne,
2000
), we think that it is unlikely the mechanism for UNC-71
because of the absence of an active protease site and the predominant
association of mutations in the domains involved in cell adhesion. Different
from the longitudinal elongation and commissural branching that depend on the
interactions between the D neuron growth cones with epidermis and other
neurons, the commissures migrate through a solely epidermal substrate. We
therefore propose that UNC-71 expressed from the epidermal cells modifies the
extracellular matrix to provide an independent, short-range, permissive
guidance cue for the D growth cones.
Our expression and functional studies show that UNC-71 appears to be
produced globally from multiple sources. It is therefore surprising to find
that unc-71 has a striking differential effect on the axon guidance
of neurons that migrate along similar path. For example, the circumferential
guidance of DA and DB neurons uses similar guidance information provided by
the UNC-6 repulsion pathway (Hedgecock et
al., 1990), and ina-1 modulates longitudinal axon
fasciculation for all ventral cord neurons
(Baum and Garriga, 1997
) (X.H.
and Y.J., unpublished). However, the DA and DB axon guidance is only
marginally affected in unc-71 single mutants, and is not any worse in
unc-71; unc-6 double mutants than unc-6 alone. Two main
differences between the type D neurons and the DA and DB neurons, first
noticed by Durbin (R. Durbin, PhD Thesis, University of Cambridge, 1987), may
account for the differential effect of unc-71. First, the type D
neurons extend their longitudinal axons immediately after the AVG neuron has
extended its ventral process down the ventral midline, whereas the DA and DB
motoneurons extend their longitudinal axons later. Second, the commissures of
the type D neurons branch out of the longitudinal axons, whereas the
commissures of the DA and DB neurons grow out directly from the cell bodies.
Our study supports a model in which UNC-71 produced by the epidermis and
neurons act as a short-range permissive cue to modulate growth cone movement
of specific classes of neurons in a temporally and spatially dynamic
manner.
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ACKNOWLEDGMENTS |
---|
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