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First published online 13 September 2006
doi: 10.1242/dev.02580
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Max-Planck-Institute for Developmental Biology, Department III/Genetics, Spemannstrasse 35, 72076 Tübingen, Germany.
* Author for correspondence (e-mail: hermann.aberle{at}tuebingen.mpg.de)
Accepted 10 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, Neuromuscular junction, Motor axon guidance, Motoneuron, Metalloprotease, Tolloid, Tolkin, Tolloid-related 1, Sidestep, Kuzbanian
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Despite the fact that motor axons initially form coherent
nerve bundles, certain growth cones leave these tightly fasciculated nerves at
specific peripheral locations in order to migrate into their target regions.
Defasciculation, the exit of axons from a nerve track, is therefore a crucial
process that needs to be strictly regulated. Failure to defasciculate at these
choice points leads to axon guidance defects and muscle innervation errors
(Araujo and Tear, 2003). Which
molecules regulate the detachment of motor axons from adherent nerve bundles?
How are choice points molecularly defined?
The Drosophila neuromuscular innervation pattern provides a
powerful experimental system to genetically dissect the molecular mechanisms
of defasciculation and motor axon guidance in vivo. Trajectories of motor
axons and their branching patterns are stereotypic and segmentally repeated
(Sink and Whitington, 1991),
as are the positions of neuromuscular terminals on muscle fibers
(Hoang and Chiba, 2001). This
relatively simple anatomy, together with the available genetic tools in
Drosophila, have been of great advantage to identify genes and gene
families with roles in motor axon guidance, including the Semaphorins
(Kolodkin et al., 1993) and
Netrins (Mitchell et al.,
1996). Furthermore, three molecules have been identified that seem
to specifically control defasciculation of motor axons: the transmembrane
tyrosine phosphatase Lar (leukocyte common antigen-related) and the members of
the immunoglobulin superfamily Beaten path (Beat) and Sidestep (Side), all
three of which give rise to highly penetrant motor axon guidance phenotypes
(Desai et al., 1996;
Fambrough and Goodman, 1996;
Krueger et al., 1996;
Sink et al., 2001). Sidestep
is expressed in somatic muscles during the period of motor axon pathfinding
and is thought to function as a muscle-derived attractant for motor nerves
(Sink et al., 2001). In
sidestep mutant embryos, motor axons frequently bypass their targets,
as they fail to defasciculate from their nerve tracts. As Lar, Beat and Side
give rise to similar bypass phenotypes when mutated, they all seem to be
involved in the defasciculation process, but how they interact
mechanistically, and whether additional regulators are required, is currently
unknown.
Here, we describe piranha, a novel axon guidance mutant with
defasciculation defects. In piranha mutants, we identified point
mutations in the evolutionarily conserved gene tolloid-related 1
(tlr1), also called tolkin (tok), which encodes an
extracellular metalloprotease (Nguyen et
al., 1994; Finelli et al.,
1995). Mutations in tlr1, but not in related
Drosophila metalloproteases, lead to stable innervation errors that
persist into larval stages. Motor nerves stay attached to each other at places
where they should diverge and consequently fail to reach their muscle targets
or use irregular routes. Genetic rescue experiments demonstrate that Tlr1
functions non-cell-autonomously, possibly in the hemolymph. In addition, the
proteolytic activity of Tlr1 is required in cooperation with other axon
guidance molecules, such as Sidestep, to terminate coherent axonaxon
interactions at choice points.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The side, tlr1 double mutants were constructed by
recombination using recessive markers of the rucuca-chromosome.
UAStlr1 and UAS-tld23A were a kind gift of
Michael O'Connor. The tolloid alleles tld10E95,
tld9B66, tld6B69 and tld7074
and Heat shock-Gal4 were provided by the Tübingen stock collection.
tlr11, tlr13, kuzE29,
kuzK01403, Mmp1k04809,
Mmp2k00604 and Mmp2KG01263,
UAS-kuzF, UAS-dsRed2 (#6282) and CG-Gal4 were obtained from
the Bloomington Stock Center
(http://flystocks.bio.indiana.edu/).
To assess neuromuscular phenotypes, mutants were crossed into the CD8-GFP-Sh
background (Zito et al.,
1999). The following Gal4-lines were used: Elav-Gal4, 24B-Gal4
(gifts of C. S. Goodman), G14-Gal4 (Aberle
et al., 2002), Cha-Gal4
(Salvaterra and Kitamoto,
2001), Serpent-Gal4 (gift of R. Reuter), PPL-Gal4 (gift of M.J.
Pankratz) and OK371-Gal4 (Mahr and Aberle,
2006). As wild-type control strains, y w1 or w;;
CD8-GFP-Sh were used. Genotypes of larvae with transgenically labeled NMJs and
motor nerves were OK371-Gal4/+; CD8-GFP-Sh,UAS-dsRed2/+ and
OK371-Gal4/+;
tlr1K788/tlr1D427,UAS-dsRed2.
Rescue crosses were performed according to the following scheme:
UASGeneX; tlr1D427/TM6B crossed to
Tissue-specific-Gal4; tlr1K788/TM6B. For heat shock
experiments, flies were allowed to lay eggs into the food of their vials for 3
hours. Embryos were then stored for 3, 6 or 9 hours or overnight at room
temperature before a heat shock (1 hour, 37°C water bath) was applied.
Complementation and mapping
Mutant lines with similar phenotypes were crossed to each other and judged
for non-complementation by the presence of mislocalized NMJs. Mutations were
mapped by meiotic recombination against the multiply marked rucucachromosome
(Bloomington). Mapping was refined using available deficiencies.
Df(3R)crb87-4, Df(3R)crb-F89-4 and Df(3R)96B did complement but Df(3R)crb87-5,
Df(3R)slo3, Df(3R)XS and Df(3R)XTA1 failed to complement tlr1
mutations. Database searches were performed at FlyBase
(http://flybase.bio.indiana.edu/)
and NCBI
(http://www.ncbi.nlm.nih.gov/).
Quantification of axon guidance phenotypes
The innervation phenotypes were quantified in intact third instar
CD8-GFP-Sh larvae. The locations of NMJs were evaluated through the
translucent cuticle in abdominal segments A2-A7 using confocal microscopy.
Embryonic guidance phenotypes were quantified in dissected embryos stained
with anti-Fas II.
Molecular analysis
Genomic DNA of the tlr1 alleles was isolated using the QIAamp DNA
Mini Kit (Qiagen), amplified by PCR and sequenced on both strands with the
BigDye Terminator kit (PE Applied Biosystems). Sequences were analyzed using
the Lasergene software package (DNAStar). The partial tlr1 cDNA clone
RH04849 was obtained from BDGP
(http://www.fruitfly.org/).
Digoxigenin-labeled sense and antisense probes for in situ hybridizations were
synthesized from RH04849 (Tautz and
Pfeifle, 1989). In situ hybridizations were imaged with an
Axiophot light microscope (Zeiss) equipped with a CCD-camera
(ProgRes3012).
Immunohistochemistry
Embryos were stained as described (Mahr
and Aberle, 2006), except that fluorescent labeling was performed
with the TSA Cyanine-3 System (Perkin Elmer). Stained embryos were dissected
with sharpened tungsten needles on microscope slides and imaged with an LSM510
confocal microscope (Zeiss) in the Cy3- and DIC channels. Signals in the
Cy3-channel were depicted in black to enhance the contrast in overlays with
the DIC images. Wandering third instar larvae were dissected in PBS on
Sylgaard plates (Dow Corning Corporation) using spring scissors (Fine Science
Tools) and insect pins (Emil Arlt). Larval fillets were fixed in 3.7%
formaldehyde/PBS for 15 minutes, washed with PTx (PBS containing 0.1% Triton
X-100) and blocked in PTx/5% normal goat serum. Primary antibodies were added
overnight at 4°C. Fillets were washed with PTx and incubated with
fluorescence-labeled secondary antibodies for 1 hour. Stained fillets were
cleared in 70% glycerol/PBS and mounted onto microscope slides. The dilutions
of the primary antibodies (supernatants) were as follows: mouse anti-Discs
large (4F3) 1:100 and mouse anti-Fasciclin II (1D4) 1:5 (gifts of C. S.
Goodman). The Cy3-conjugated secondary antibodies were diluted 1:400
(Molecular Probes or Jackson ImmunoResearch). Fluorescently-labeled larvae
were examined with a TCS SPL (Leica) or LSM510 (Zeiss) confocal laser scanning
microscope. Projections and single images were adjusted for brightness and
contrast using Adobe Photoshop.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In
each abdominal hemisegment, two major motor nerves defasciculate into five
nerve branches (Fig. 1A). The
segmental nerve (SN) bifurcates into the SNa and SNc, and the intersegmental
nerve gives rise to a dorsal branch (ISN) and two ventral branches (ISNb and
ISNd).
In a large-scale mutagenesis screen for genes that affect the structure and
maintenance of NMJs (Parnas et al.,
2001; Aberle et al.,
2002), we discovered four alleles of a novel mutant,
piranha, which showed missing and/or mislocalized NMJs. To visualize
NMJs in piranha mutants, we used the transgenically encoded fusion
protein CD8-GFP-Sh (Zito et al.,
1999). CD8-GFP-Sh is controlled by a muscle-specific promotor and
consists of the extracellular and transmembrane domain of human CD8, fused
cytoplasmatically to GFP and a C-terminal domain of the Shaker potassium
channel. It binds to the postsynaptic protein Discs large (Dlg) and highlights
all NMJs consisting of type Ib and Is boutons
(Zito et al., 1999). Using
this synaptic marker, we could evaluate NMJs through the translucent cuticle
of intact third instar larvae.
In wild-type larvae, the dorsalmost NMJs on muscle pairs 1/9 and 2/10 are normally arranged symmetrically relative to the axis of the ISN (Fig. 1A-C). In piranhaD427/piranhaK788 mutants, however, we found that muscles 1 and 9 either lacked NMJs completely or were innervated at ectopic sites in 38.1% (muscle 1) or 25.0% (muscle 9) of hemisegments (Table 1, Fig. 1D). We observed even stronger defects in ventral muscle regions. In wild type, the ISNb defasciculates from the ISN and forms, among others, NMJs in the cleft between muscles 6/7 and on a ventral and medial position on muscles 12 and 13, respectively (Fig. 1E,F). In piranha mutants, we found that NMJs on muscle 12 and 13 were mislocalized or absent in 64.3 and 56.0% of hemisegments, respectively (Table 1, Fig. 1G). Muscles 6 and 7 showed an erroneous innervation pattern in 23.9 and 34.6% of hemisegments. It is important to note that the pattern and degree of misinnervation varied between hemisegments along the anterior-posterior axis and also between two hemisegments located on contralateral sides. We determined the total number of misinnervated muscles per hemisegment and found that between 7.1 and 46.4% of muscles were affected in piranha mutant hemisegments (n=84) compared with 0-14.3% in wild-type larvae (n=44). Thus, innervation errors occurred in all body wall regions and in every hemisegment, but the innervation pattern was unique for each hemisegment, indicating that the innervation process is largely autonomous within a given hemisegment.
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). We did not observe neuromuscular Dlg staining in places
where CD8-GFP-Sh clusters were absent, neither in wild-type
(Fig. 1H-J) nor in
piranha mutant larvae (Fig.
1K-M). CD8-Sh-GFP therefore faithfully reflects the presence and
positions of NMJs.
|
). In
wildtype larvae, muscles 12 and 13 are innervated by a ventrodorsal projection
of the ISNb pathway (Fig.
2A-C). In piranha mutants, we found that muscles 12 and
13 were often innervated via a dorsoventral projection of the ISN or SNa,
which induced the formation of NMJs at ectopic sites
(Fig. 2D-F). This suggests that
motoneuronal growth cones of the ISNb pathway failed to navigate into the
ventral muscle field via their normal route. Despite this initial failure,
however, some growth cones eventually managed to detach from the ISN or SNa
and innervated muscles 12 and 13 from the dorsal side (reach back phenotypes
via a different nerve route). These experiments show that motor axons are
misrouted in piranha mutants, which leads to the formation of
synaptic terminals at ectopic locations.
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), we
expressed the Discosoma Red Fluorescent Protein (UAS-dsRed2)
specifically in motoneurons (OK371-Gal4) in the background of CD8-GFP-Sh
wild-type or piranha mutant animals. Motor axons and presynaptic
terminals were therefore highlighted in red and muscles and postsynaptic
terminals in green (Fig. 2G-L).
In wildtype larvae, the SN and ISN nerves form a single nerve bundle that
defasciculates into five nerve branches at the ventralmost choice point (shown
schematically in Fig. 2G). The
ISNb and ISNd detach from the ISN, whereas the SNc detaches from the SNa
(Fig. 2J). In piranha
mutants, we observed that the ISNb frequently stayed attached to the ISN,
sometimes as a separately identifiable axon bundle, and reached its target
muscles via irregular routes (Fig.
2H,K). We found similar defasciculation errors in the SN pathway.
As shown in Fig. 2I,L, the SN
nerve often grew too far to the posterior, because the SNa defasciculates much
too late. piranha mutant larvae display, therefore, severe bypass and
misrouting phenotypes, primarily due to motor axons remaining attached to each
other at choice points.
Guidance errors in piranha mutants occur at embryonic stages
We asked next at which time point these projection errors develop, and we
examined motoneuronal trajectories during stages 16-17 of embryonic
development, when neuromuscular connectivity is established. The ISN of
wild-type embryos has three clearly visible branch points
(Fig. 3A). In piranha
mutants, we observed in 84.4% of hemisegments
(Table 1) that the branch
points appeared underdeveloped and/or the ISN was stalled at the second branch
point and barely reached the position of the third branch point (arrow in
Fig. 3B). Within the developing
SNa pathway, axons normally bifurcate into a dorsal and posterior branch at
the dorsal edge of muscle 12 (Fig.
3C). In piranha mutant embryos, we found SNa defects in
51.2% of hemisegments, including, for example, two dorsal branches of the SNa
(arrowheads in Fig. 3D). In
ventral muscle regions, the ISNb failed to defasciculate from the ISN in 96.2%
of hemisegments. The ISNb was either tightly attached to the ISN (fusion
bypass, see left segment in Fig.
3D and Table 1) or
formed a separated parallel nerve bundle (split bypass, middle and right
segment in Fig. 3D and
Table 1). In addition, we and
others (M. Serpe and M. O'Connor, personal communication) observed
defasciculation defects in Fasciclin II-positive nerve tracts in the embryonic
central nervous system (data not shown). The innervation defects do not
strongly compromise embryogenesis and larval development, because we recover
piranhaD427 mutant second and early third instar larvae at
the expected ratios (data not shown). Based on these observations, we conclude
that the piranha mutant guidance phenotype arises during
embryogenesis, and the defects are quantitatively stronger but qualitatively
similar to the larval defects.
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; Finelli et al.,
1995). To examine if piranha is allelic to tlr1,
we performed complementation crosses using existing tlr1 alleles.
Both tlr11 and tlr13 did not
complement our piranha alleles, and the transheterozygous animals had
both axon guidance and posterior crossvein defects. The tlr1 cDNA
encompasses 5.4 kb and encodes a predicted extracellular metalloprotease
(Nguyen et al., 1994;
Finelli et al., 1995).
Consisting of 1464 amino acids, Tlr1 is composed of an N-terminal prodomain, a
central metalloprotease domain and a C-terminal protein-protein interaction
domain containing five CUB (complement subcomponents C1r/C1s, Uegf, BMP-1) and
two EGF (epidermal growth factor) domains
(Fig. 4B).
To identify potential molecular lesions, we sequenced the Tlr1-encoding
exons and found single point mutations in all four piranha alleles.
We therefore renamed our piranha alleles to
tlr1K788, tlr1D427,
tlr1K598 and tlr1I678. Three of them
contain nonsense mutations in the N-terminal prodomain: R409Stop in
tlr1K788, Q464Stop in tlrD427 and
Q472Stop in tlr1K598
(Fig. 4B). Provided that the
prodomain functions only in inhibiting the activity of the protease domain,
these alleles are probably null alleles. Consistent with this assumption is
the previous classification of tlr11, which contains a
stop codon in the protease domain, as a null allele
(Finelli et al., 1995). In the
fourth allele, we identified a serine to threonine exchange within the
protease domain (S669T in tlr1I678). This highly conserved
serine residue is located within the sequence motif `SIMHY', which forms a
characteristic loop, the Met-turn, that positions the terminal tyrosine
residue to coordinate the zinc ion in the active site
(Gomis-Ruth et al., 1993).
Insufficient zinc coordination probably renders the protease domain
non-functional in tlr1I678. Indeed, a point mutation in
the very same serine residue of the related metalloprotease Tolloid (S276F in
tolloid10E) has been isolated in a screen for new
tolloid alleles (Finelli et al.,
1994).
tolloid (tld) is a close sequence homolog and genomic
neighbor of tlr1, with less than 1000 base pairs separating the two
genes (Fig. 4A). Tolloid is
involved in the determination of the dorsoventral body axis and functions as
an activator of Decapentaplegic (Dpp) signaling in dorsal regions of
blastoderm embryos (Shimell et al.,
1991; Marques et al.,
1997). In mammals, four relatives of Tlr1 and Tld have been
identified (Fig. 4B): mammalian
Tolloid (Tld), bone morphogenetic protein 1 (Bmp1), Tolloid-like 1 (Tll1) and
Tolloid-like 2 (Tll2), the first two of which are isoforms derived from the
same gene (Takahara et al.,
1994; Takahara et al.,
1996; Scott et al.,
1999). The metalloprotease domain of Drosophila Tlr1 is
64.5, 67.5 and 67.0% identical to its mouse relatives Tld, Tll1 and Tll2,
respectively. The CUB/EGF protein-protein interaction domain is also well
conserved and shows 42.0, 42.5 and 43.9% identity to these proteins.
|
;
Finelli et al., 1995). We
observed tlr1 expression first in the dorsal blastoderm
(Fig. 4C). In the extended germ
band stage, it was transcribed in progenitor cells of the visceral mesoderm
(Fig. 4D). At stages 13-15,
tlr1 was expressed in somatic muscles and the midgut
(Fig. 4E). Beginning with stage
15, tlr1 transcripts also appeared in a few cells in the ventral
nerve cord, the thoracic peripheral nervous system and the ring gland
(Fig. 4F). Interestingly,
tlr1 was strongly expressed in muscles but not in neurons when motor
axons leave the central nervous system, indicating that muscle-derived Tlr1
may be important for motor axon guidance.
Transgenic rescue experiments reveal that Tlr1 functions non-cell-autonomously
To determine in which tissue and during which developmental period Tlr1 is
required, we performed transgenic rescue experiments using the Gal4/UAS
system. We induced transcription of wild-type Tlr1 in a tlr1 mutant
background during different developmental stages using Heat shock-Gal4.
Compared with wildtype animals (Fig.
5A), mutant animals transgenic for UAS-tlr1 and Heat
shock-Gal4 displayed strong innervation defects when reared at room
temperature, because expression of Tlr1 is not induced
(Fig. 5B). By contrast, mutant
embryos that received a 1-hour heat shock (37°C) when they were 3-6, 6-9
or 9-12 hours old (stages 6-9, 10-12 or 12-15, respectively) developed into
larvae with a normal innervation pattern
(Fig. 5C). When we applied the
heat shock at the end of embryogenesis (stage 17), however, we were unable to
rescue the mislocalized NMJs in tlr1 mutants, indicating that Tlr1 is
ineffective after NMJs have formed. Hence, Tlr1 functions during mid- to
late-embryonic stages, when neuromuscular connectivity is established.
To examine whether Tlr1 is required in nerves or muscles, we performed rescue experiments using tissue-specific Gal4-lines. As tlr1 is strongly expressed in muscles during motor axon pathfinding, we reasoned that muscle-specific expression of Tlr1 might be sufficient for rescue. Indeed, expression of Tlr1 in all somatic muscles using G14-Gal4 completely restored the innervation pattern in homozygous mutant larvae (Fig. 5D). The rescued larvae were indistinguishable from wild-type animals with respect to the size and localization of their NMJs. Unexpectedly, however, expression of Tlr1 in all postmitotic neurons using Elav-Gal4 also completely restored the wild-type innervation pattern (Fig. 5E). When we expressed Tlr1 only in cholinergic neurons using Cha-Gal4, the mutant phenotype was not rescued, demonstrating that UAS-tlr1 is not unspecifically expressed in the absence of a Gal4 driver (Fig. 5F). From these results, it appeared that Tlr1 could be expressed either in muscles or all neurons to fully rescue the guidance defects. As Tlr1 is likely to be a secreted protease, this finding could be explained by a non-cell-autonomous function of Tlr1. If secretion into the extracellular matrix or hemolymph is sufficient to deliver Tlr1 to the locations where its proteolytic activity is required, then expression of Tlr1 in tissues normally irrelevant for motor axon guidance should rescue the innervation defects. Using fat body-specific Pumpless-Gal4 (PPLGal4) or two different hemocyte-specific drivers (CG-Gal4 and Serpent-Gal4), we could completely restore the wild-type innervation pattern in tlr1 mutants (data not shown), supporting a cell non-autonomous function for Tlr1.
To test whether Tlr1 is also sufficient to misdirect motor axons in a wild-type background, we overexpressed it either in the nervous system (Elav-Gal4) or in all muscles (G14-Gal4) but we did not observe any motor axon guidance phenotypes, as visualized with CD8-GFP-Sh (data not shown). We noticed, however, that overexpression of Tlr1 in wing discs (24B-Gal4) leads to a large central wing blister, suggesting that the function of Tlr1 during wing development is dosage sensitive: too little Tlr1 leads to missing posterior crossveins, whereas too much Tlr1 causes loss of adhesion between wing epithelia (data not shown).
The metalloproteases Tolloid and Kuzbanian cannot functionally replace Tlr1
As mutations in tlr1 affect a large subset, but not all, NMJs, we
wondered whether the proteolytic activities of other metalloproteases are
additionally required for pathway selection. As a closely related paralog of
Tlr1, Tolloid may be required for the innervation of the remaining muscles. As
strong alleles of tld are embryonic lethal, we searched for
transheterozygous allelic combinations that would survive to third instar
larvae. Compared with tlr1 mutants
(Fig. 6A), we were not able to
detect mislocalized or structurally abnormal NMJs in
tld6B69/tld7074,
tld6B69/tld9B66 or
tld9B66/tld10E95 mutants, indicating
that Tolloid is not required for wiring and maintaining NMJs
(Fig. 6B).
|
;
Stocker et al., 1995).
Kuzbanian (Kuz), a member of the ADAM family in Drosophila, is
expressed in neurons and has been shown to play a role in axon guidance at the
ventral midline, raising the possibility that it may also regulate pathfinding
of motor axons (Fambrough et al.,
1996; Schimmelpfeng et al.,
2001). When we examined
kuzE29/kuzK01403 mutant larvae,
however, all NMJs were at their wild-type positions, indicating that motor
axons project along correct trajectories
(Fig. 6C). Other prime
candidates for regulators of axonal pathfinding are matrix metalloproteases
(Mmps). Mmps in general process extracellular matrix components, which is
necessary for tissue remodeling and cell migration, but has also been shown to
be crucial for axon guidance (Webber et
al., 2002). Drosophila has only two Mmps, Mmp1 and Mmp2.
During the time of motor axon pathfinding, Mmp1 is expressed in midline glial
cells and Mmp2 in neurons and mesodermal cells, all tissues that are relevant
for navigating growth cones (Llano et al.,
2000; Llano et al.,
2002; Page-McCaw et al.,
2003). When we analyzed Mmp1k04809,
Mmp2k00604 and Mmp2KG01263 mutant
larvae, however, we failed to detect any neuromuscular abnormalities (data not
shown). We next examined if Tlr1 and Tld could functionally replace each other if expressed in similar tissues. To test for functional redundancy, we crossed UAS-tld or UAS-tlr1 into the tlr1 mutant background and expressed either gene with Elav-Gal4 or 24B-Gal4. In contrast to experiments using UAS-tlr1 (Fig. 6D), Tld was not able to substantially rescue the tlr1 mutant phenotype, irrespective of whether we expressed it in neurons or muscles (Fig. 6E). Similarly, UAS-kuz failed to improve the projections errors in tlr1 mutants (Fig. 6F), showing that neither Tld nor Kuz could functionally replace Tlr1. Thus, loss- and gain-of-function analysis of metalloproteases belonging to the matrixin, adamlysin and astacin families support a key-regulatory role of Tlr1 in motor axon guidance in Drosophila.
Axon guidance phenotypes are strongly enhanced in tlr1,side double mutants
To identify candidate molecules that might act in concert with Tlr1, we
searched for mutants with related phenotypes. In our large-scale EMS
mutagenesis screen of the larval NMJ, we identified four new alleles of
sidestep (sideC137, sideI306,
sideI1563, sideK717). Sidestep, originally
identified by H. Sink and colleagues (Sink
et al., 2001), is a transmembrane receptor of the immunoglobulin
superfamily that functions as a muscle-derived attractant for motor axons.
Similarly to tlr1 mutants (Fig.
7A,7A'), we found missing and abnormally localized NMJs in
sideC137/sideI1563 mutant larvae, regardless of whether
we used CD8-GFP-Sh (Fig. 7B) or
anti-Fas II antibodies (Fig.
7B') as detection methods. The immunohistochemical stainings also
confirmed that the CD8-GFP-Sh marker reliably reflected the axon guidance
defects in side mutants. In side mutant embryos, pathfinding
of the ISNb was defective in 83.8% of hemisegments
(Table 1). tlr1 and
side mutants therefore share a variety of phenotypic similarities:
(1) embryonic defasciculation defects affect all pathways and persist into
larval stages; (2) failures to reach final branch points; (3) innervation
patterns vary between segments; (4) ventral muscles are more frequently
affected; and (5) NMJs can be mislocalized or absent on an affected muscle
fiber. For a majority of muscles, however, the side mutant phenotype
was more penetrant (Fig.
7A",B").
As mutations in genes that function together in the same biological process often share phenotypic similarities, we created side,tlr1 double mutants using genetic null alleles. If Tlr1 and Side function in a linear pathway, then double mutants would be predicted to exhibit a phenotype that resembles each single mutant phenotype. In tlr1D427,sideC137/tlr1K788,sideC137 double mutants, however, we observed a strong enhancement of each single mutant phenotype. Double mutant larvae lacked virtually all NMJs on all ventral muscles in all abdominal hemisegments (Fig. 7C,7C', Table 1). Muscles located in lateral or dorsal regions of the body wall also showed an increase in innervation errors, albeit less dramatically (Fig. 7C", Table 1). The increase in phenotypic strength suggests that tlr1 and side function in parallel pathways, or that their functions converge on shared components further downstream. Interestingly, tlr1,side double mutants were still capable of forming NMJs in lateral and dorsal regions, suggesting that additional gene products are involved in ensuring complete innervation of the musculature.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Sink et al., 2001;
de Jong et al., 2005). Apart
from embryonic lethality, a possible reason is presumably that the detailed
analysis of larval motor axon phenotypes is hampered by the availability of
suitable methods other than manual dissection. We used transgenic methods for
the visualization of the neuromuscular wiring pattern in intact larvae. The
fluorescent CD8-GFP-Sh marker specifically highlights the postsynaptic
apparatus of each muscle fiber, revealing the structure and location of each
NMJ (Zito et al., 1999). As
CD8-GFPSh is under the control of the myosin heavy chain promotor, we combined
it with the Gal4/UAS-system to simultaneously express the red fluorescent
protein dsRed2 in motoneurons. CD8-GFP-Sh by itself indirectly reflects the
projection pattern of motor axons by reliably revealing the location of each
NMJ. However, the combination of both CD8-GFP-Sh and motoneuron-specific
dsRed2 highlights the entire neuromuscular connectivity pattern in living
animals.
Supported by these non-invasive methods, we show that tlr1 mutants
have defects in motor axon guidance rather than in target recognition or
innervation site selection. First, motor nerves show defasciculation defects
already during embryogenesis, even before they reach their target muscles.
Second, motor axons remain physically attached to each other beyond their
destined branch points and until late larval stages. Third, the bypass, stall
and misrouting phenotypes are similar to those found in sidestep
mutants, a known regulator of motor axon guidance
(Sink et al., 2001). As a
result, motor axons fail to reach their targets in tlr1 mutants or do
so from unusual directions, which leads to anomalous muscle innervation. The
formation of NMJs at ectopic locations suggests that the growth direction and
entry point of the motor nerve may instruct the final location of an NMJ. Even
if the position of an NMJ is predetermined by the muscle fiber, the incoming
nerve terminal appears to be able to relocate postsynaptic components.
In tlr1 mutants, all abdominal segments show innervation defects,
and all pathways are affected. The intrasegmental phenotype, however, is quite
variable, i.e. the innervation pattern of a given hemisegment differs visibly
from the adjacent or contralateral hemisegment, indicating that neuromuscular
wiring is largely autonomous for a given hemisegment. The variability further
implies that Tlr1 may not regulate specific and invariable guidance decisions,
but rather plays a general role in defasciculation. Migrating growth cones in
tlr1 mutants apparently do not strictly rely on single molecular
labels positioned at specific locations. These conclusions support the idea
that exact target selection appears to be a rather stochastic process, in
which the growth cones integrate attractive and repulsive cues provided by the
microenvironment of surrounding cells
(Winberg et al., 1998).
How does Tlr1 function?
Metalloproteases have been implicated in a variety of cellular processes,
including cell migration, angiogenesis and metastasis
(Sternlicht and Werb, 2001;
Yong et al., 2001). Neuronal
growth cones migrate through an environment that is rich in different
extracellular surfaces and may thus exploit similar molecules and mechanisms
as migrating cells. Despite the wealth of data on cell migration, only a few
reports have been published implicating metalloproteases in axon outgrowth and
guidance (Fambrough et al.,
1996; Galko and
Tessier-Lavigne, 2000; Hattori
et al., 2000; Schimmelpfeng et
al., 2001; Webber et al.,
2002; McFarlane,
2003; Vaillant et al.,
2003; VanSaun et al.,
2003; Hehr et al.,
2005; Jaworski et al.,
2006). With regard to motor axons, only ADM-1 (unc-71), a
member of the ADAM family in Caenorhabditis elegans, has been shown
to regulate pathfinding (Huang et al.,
2003). Tlr1 belongs to the astacin family of metalloproteases and
is highly related to Drosophila Tolloid. Despite this high degree of
conservation, we and others have shown that these two proteins have mutually
exclusive functions (Nguyen et al.,
1994; Serpe et al.,
2005). While Tld cannot functionally replace Tlr1, it is still
possible that other metalloproteases with redundant functions assist Tlr1 in
defasciculation control, because not all guidance decisions are affected in
tlr1 mutants. Our loss- and gain-of-function analysis of related
metalloproteases, however, did not support this possibility. In addition, no
other metalloprotease has so far been recovered from mutant screens
(van Vactor et al., 1993;
Kraut et al., 2001;
Sink et al., 2001). These
observations support the idea that Tlr1 may be a key regulatory member of the
metalloprotease family in Drosophila that controls motor axon
guidance.
As a secreted metalloprotease, Tlr1 is predicted to function
extracellularly, either in the extracellular matrix or in the interstitial
fluid. Consistent with this prediction, overexpression of Tlr1 in hemocytes or
cells of the fat body could fully rescue the tlr1 mutant phenotype.
Neither hemocytes nor fat-storing cells have so far been implicated in axon
guidance. Hence, it is unlikely that Tlr1 remained associated with the
extracellular matrix of these cells, but probably got released into the
hemolymph. The circulating hemolymph then distributed it to where it was
required. As endogenous Tlr1 is expressed in developing muscles during the
period of axonal pathfinding, it is possibly secreted from there into the
hemolymph to either proteolytically activate a repellent on motor nerves to
induce defasciculation or to activate an attractant on muscles. The axon
guidance receptor Sidestep is expressed on muscles and functions as an
attractant for motor axons (Sink et al.,
2001). Based on similarities in their loss-of-function phenotypes,
Tlr1 could be required to activate the attractive function of Side. The
phenotype of tlr1,side double mutants, however, was clearly stronger
compared with each single mutant, indicating that they regulate the same
biological process but that they function either independently or that the two
functions converge further downstream. Tlr1 could therefore regulate the
activity of an alternative pathway. Interestingly, M. O`Connor's group
suggested that Tlr1 regulates motor axon guidance in part by activating latent
TGF-ß ligands (M. Serpe and M. O'Connor, unpublished). Although the exact
molecular function of Tlr1 is currently not known, the data presented here
clearly demonstrate that the evolutionarily conserved metalloprotease
Tolloid-related 1 is necessary for motor axon guidance in
Drosophila.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aberle, H., Haghighi, A. P., Fetter, R. D., McCabe, B. D., Magalhaes, T. R. and Goodman, C. S. (2002). wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila. Neuron 33,545 -558.[CrossRef][Medline]
Araujo, S. J. and Tear, G. (2003). Axon guidance mechanisms and molecules: lessons from invertebrates. Nat. Rev. Neurosci. 4,910 -922.[CrossRef][Medline]
Bond, J. S. and Beynon, R. J. (1995). The astacin family of metalloendopeptidases. Protein Sci. 4,1247 -1261.[Medline]
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118,401 -415.[Abstract]
Budnik, V., Koh, Y. H., Guan, B., Hartmann, B., Hough, C., Woods, D. and Gorczyca, M. (1996). Regulation of synapse structure and function by the Drosophila tumor suppressor gene dlg. Neuron 17,627 -640.[CrossRef][Medline]
de Jong, S., Cavallo, J. A., Rios, C. D., Dworak, H. A. and Sink, H. (2005). Target recognition and synaptogenesis by motor axons: responses to the sidestep protein. Int. J. Dev. Neurosci. 23,397 -410.[CrossRef][Medline]
Desai, C. J., Gindhart, J. G., Jr, Goldstein, L. S. and Zinn, K. (1996). Receptor tyrosine phosphatases are required for motor axon guidance in the Drosophila embryo. Cell 84,599 -609.[CrossRef][Medline]
Fambrough, D. and Goodman, C. S. (1996). The Drosophila beaten path gene encodes a novel secreted protein that regulates defasciculation at motor axon choice points. Cell 87,1049 -1058.[CrossRef][Medline]
Fambrough, D., Pan, D., Rubin, G. M. and Goodman, C. S.
(1996). The cell surface metalloprotease/disintegrin Kuzbanian is
required for axonal extension in Drosophila. Proc. Natl. Acad. Sci.
USA 93,13233
-13238.
Finelli, A. L., Bossie, C. A., Xie, T. and Padgett, R. W. (1994). Mutational analysis of the Drosophila tolloid gene, a human BMP-1 homolog. Development 120,861 -870.[Abstract]
Finelli, A. L., Xie, T., Bossie, C. A., Blackman, R. K. and Padgett, R. W. (1995). The tolkin gene is a tolloid/BMP-1 homologue that is essential for Drosophila development. Genetics 141,271 -281.[Abstract]
Galko, M. J. and Tessier-Lavigne, M. (2000).
Function of an axonal chemoattractant modulated by metalloprotease activity.
Science 289,1365
-1367.
Gomis-Ruth, F. X., Stocker, W., Huber, R., Zwilling, R. and Bode, W. (1993). Refined 1.8 A X-ray crystal structure of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L. Structure determination, refinement, molecular structure and comparison with thermolysin. J. Mol. Biol. 229,945 -968.[CrossRef][Medline]
Hattori, M., Osterfield, M. and Flanagan, J. G.
(2000). Regulated cleavage of a contact-mediated axon repellent.
Science 289,1360
-1365.
Hehr, C. L., Hocking, J. C. and McFarlane, S.
(2005). Matrix metalloproteinases are required for retinal
ganglion cell axon guidance at select decision points.
Development 132,3371
-3379.
Hoang, B. and Chiba, A. (2001). Single-cell analysis of Drosophila larval neuromuscular synapses. Dev. Biol. 229,55 -70.[CrossRef][Medline]
Huang, X., Huang, P., Robinson, M. K., Stern, M. J. and Jin,
Y. (2003). UNC-71, a disintegrin and metalloprotease (ADAM)
protein, regulates motor axon guidance and sex myoblast migration in C.
elegans. Development
130,3147
-3161.
Jaworski, D. M., Soloway, P., Caterina, J. and Falls, W. A. (2006). Tissue inhibitor of metalloproteinase-2(TIMP-2)-deficient mice display motor deficits. J. Neurobiol. 66, 82-94.[CrossRef][Medline]
Kolodkin, A. L., Matthes, D. J. and Goodman, C. S. (1993). The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 75,1389 -1399.[CrossRef][Medline]
Kraut, R., Menon, K. and Zinn, K. (2001). A gain-of-function screen for genes controlling motor axon guidance and synaptogenesis in Drosophila. Curr. Biol. 11,417 -430.[CrossRef][Medline]
Krueger, N. X., Van Vactor, D., Wan, H. I., Gelbart, W. M., Goodman, C. S. and Saito, H. (1996). The transmembrane tyrosine phosphatase DLAR controls motor axon guidance in Drosophila. Cell 84,611 -622.[CrossRef][Medline]
Llano, E., Pendas, A. M., Aza-Blanc, P., Kornberg, T. B. and
Lopez-Otin, C. (2000). Dm1-MMP, a matrix metalloproteinase
from Drosophila with a potential role in extracellular matrix remodeling
during neural development. J. Biol. Chem.
275,35978
-35985.
Llano, E., Adam, G., Pendas, A. M., Quesada, V., Sanchez, L. M.,
Santamaria, I., Noselli, S. and Lopez-Otin, C. (2002).
Structural and enzymatic characterization of Drosophila Dm2-MMP, a
membrane-bound matrix metalloproteinase with tissue-specific expression.
J. Biol. Chem. 277,23321
-23329.
Mahr, A. and Aberle, H. (2006). The expression pattern of the Drosophila vesicular glutamate transporter: A marker protein for motoneurons and glutamatergic centers in the brain. Gene Expr. Patterns 6,209 -309.
Marques, G., Musacchio, M., Shimell, M. J., Wunnenberg-Stapleton, K., Cho, K. W. and O'Connor, M. B. (1997). Production of a DPP activity gradient in the early Drosophila embryo through the opposing actions of the SOG and TLD proteins. Cell 91,417 -426.[CrossRef][Medline]
McFarlane, S. (2003). Metalloproteases: carving out a role in axon guidance. Neuron 37,559 -562.[CrossRef][Medline]
Mitchell, K. J., Doyle, J. L., Serafini, T., Kennedy, T. E., Tessier-Lavigne, M., Goodman, C. S. and Dickson, B. J. (1996). Genetic analysis of Netrin genes in Drosophila: Netrins guide CNS commissural axons and peripheral motor axons. Neuron 17,203 -215.[CrossRef][Medline]
Nguyen, T., Jamal, J., Shimell, M. J., Arora, K. and O'Connor, M. B. (1994). Characterization of tolloid-related-1: a BMP-1-like product that is required during larval and pupal stages of Drosophila development. Dev. Biol. 166,569 -586.[CrossRef][Medline]
Nose, A., Takeichi, M. and Goodman, C. S. (1994). Ectopic expression of connectin reveals a repulsive function during growth cone guidance and synapse formation. Neuron 13,525 -539.[CrossRef][Medline]
Page-McCaw, A., Serano, J., Sante, J. M. and Rubin, G. M. (2003). Drosophila matrix metalloproteinases are required for tissue remodeling, but not embryonic development. Dev. Cell 4,95 -106.[CrossRef][Medline]
Parnas, D., Haghighi, A. P., Fetter, R. D., Kim, S. W. and Goodman, C. S. (2001). Regulation of postsynaptic structure and protein localization by the Rhotype guanine nucleotide exchange factor dPix. Neuron 32,415 -424.[CrossRef][Medline]
Salvaterra, P. M. and Kitamoto, T. (2001). Drosophila cholinergic neurons and processes visualized with Gal4/UAS-GFP. Gene Expr. Patterns 1,73 -82.[CrossRef][Medline]
Schimmelpfeng, K., Gogel, S. and Klämbt, C. (2001). The function of leak and kuzbanian during growth cone and cell migration. Mech. Dev. 106, 25-36.[CrossRef][Medline]
Scott, I. C., Blitz, I. L., Pappano, W. N., Imamura, Y., Clark, T. G., Steiglitz, B. M., Thomas, C. L., Maas, S. A., Takahara, K., Cho, K. W. et al. (1999). Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis. Dev. Biol. 213,283 -300.[CrossRef][Medline]
Serpe, M., Ralston, A., Blair, S. S. and O'Connor, M. B.
(2005). Matching catalytic activity to developmental function:
tolloid-related processes Sog in order to help specify the posterior crossvein
in the Drosophila wing. Development
132,2645
-2656.
Shimell, M. J., Ferguson, E. L., Childs, S. R. and O'Connor, M. B. (1991). The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein 1. Cell 67,469 -481.[CrossRef][Medline]
Sink, H. and Whitington, P. M. (1991). Location and connectivity of abdominal motoneurons in the embryo and larva of Drosophila melanogaster. J. Neurobiol. 22,298 -311.[CrossRef][Medline]
Sink, H., Rehm, E. J., Richstone, L., Bulls, Y. M. and Goodman, C. S. (2001). sidestep encodes a target-derived attractant essential for motor axon guidance in Drosophila. Cell 105, 57-67.[CrossRef][Medline]
Sternlicht, M. D. and Werb, Z. (2001). How matrix metalloproteinases regulate cell behavior. Annu. Rev. Cell Dev. Biol. 17,463 -516.[CrossRef][Medline]
Stocker, W., Grams, F., Baumann, U., Reinemer, P., Gomis-Ruth, F. X., McKay, D. B. and Bode, W. (1995). The metzincins-topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases. Protein Sci. 4, 823-840.[Medline]
Takahara, K., Lyons, G. E. and Greenspan, D. S.
(1994). Bone morphogenetic protein-1 and a mammalian tolloid
homologue (mTld) are encoded by alternatively spliced transcripts which are
differentially expressed in some tissues. J. Biol.
Chem. 269,32572
-32578.
Takahara, K., Brevard, R., Hoffman, G. G., Suzuki, N. and Greenspan, D. S. (1996). Characterization of a novel gene product (mammalian tolloid-like) with high sequence similarity to mammalian tolloid/bone morphogenetic protein-1. Genomics 34,157 -165.[CrossRef][Medline]
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[CrossRef][Medline]
Tessier-Lavigne, M. and Goodman, C. S. (1996).
The molecular biology of axon guidance. Science
274,1123
-1133.
Vaillant, C., Meissirel, C., Mutin, M., Belin, M. F., Lund, L. R. and Thomasset, N. (2003). MMP-9 deficiency affects axonal outgrowth, migration, and apoptosis in the developing cerebellum. Mol. Cell. Neurosci. 24,395 -408.[CrossRef][Medline]
van Vactor, D., Sink, H., Fambrough, D., Tsoo, R. and Goodman, C. S. (1993). Genes that control neuromuscular specificity in Drosophila. Cell 73,1137 -1153.[CrossRef][Medline]
VanSaun, M., Herrera, A. A. and Werle, M. J. (2003). Structural alterations at the neuromuscular junctions of matrix metalloproteinase 3 null mutant mice. J. Neurocytol. 32,1129 -1142.[CrossRef][Medline]
Webber, C. A., Hocking, J. C., Yong, V. W., Stange, C. L. and
McFarlane, S. (2002). Metalloproteases and guidance of
retinal axons in the developing visual system. J.
Neurosci. 22,8091
-8100.
Winberg, M. L., Mitchell, K. J. and Goodman, C. S. (1998). Genetic analysis of the mechanisms controlling target selection: complementary and combinatorial functions of netrins, semaphorins, and IgCAMs. Cell 93,581 -591.[CrossRef][Medline]
Yong, V. W., Power, C., Forsyth, P. and Edwards, D. R. (2001). Metalloproteinases in biology and pathology of the nervous system. Nat. Rev. Neurosci. 2, 502-511.[CrossRef][Medline]
Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y. and Goodman, C. S. (1999). Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila. Neuron 22,719 -729.[CrossRef][Medline]
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