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First published online 22 February 2006
doi: 10.1242/dev.02300
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Department of Medical Genetics and Microbiology, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, M5S 1A, Canada.
* Author for correspondence (e-mail: peter.roy{at}utoronto.ca)
Accepted 26 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Muscle arms, Muscle development, Elegans, FGF, RNAi, Integrin
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This activity is often hijacked in cancerous cells to
promote neo-vascularization of tumors
(Folkman, 1971;
Ogawa et al., 2000;
Plate et al., 1992) and the
metastatic colonization of healthy tissue
(Keilhack et al., 2001). Many
of these migrations and cell extensions are regulated by well characterized
guidance cues, cell-surface receptors and downstream signaling elements that
function together to control cytoskeletal reorganization
(Dickson, 2002;
Pollard and Borisy, 2003).
However, the components that guide the migration and cell extension of other
cell types remain a mystery (Rorth,
2003).
Intriguingly, developing muscles in a variety of animals also extend plasma
membrane in a regulated fashion. At the sites of incoming motor axons, muscle
membrane extensions called microspikes or myopodia have been observed in
Drosophila (Bate,
1990; Ritzenthaler et al.,
2000), Xenopus
(Kullberg et al., 1977), rat
(Uhm et al., 2001) and mouse
(Misgeld et al., 2002), and
probably facilitate the development of the neuromuscular junction (NMJ). Mouse
myopodia are enriched with clusters of acetylcholine receptors (AChRs)
(Misgeld et al., 2002),
whereas in rat, agrin can elicit the microspikes and mimic the effects of
incoming axons (Uhm et al.,
2001). Although agrin is well known to activate the MuSK receptor
tyrosine kinase and thereby promote clustering of AChRs at the postsynaptic
membrane (DeChiara et al.,
1996; Gautam et al.,
1996), it is unknown if agrin elicits muscle membrane extension
through a similar signaling pathway.
The muscles of C. elegans also extend specialized plasma membrane
to motor axons (Stretton,
1976; Sulston and Horvitz,
1977; White et al.,
1986). In wild-type adult animals, there are 95 body wall muscles
(BWMs) that are distributed along the length of the worm in four rows
(Sulston and Horvitz, 1977;
Sulston et al., 1983)
(Fig. 1). There are two dorsal
and two ventral BWM quadrants. The dorsal muscles extend membrane called
muscle arms to the dorsal nerve cord, where muscle arm termini harbor the
postsynaptic element of the NMJ (White et
al., 1976). Similarly, ventral muscles extend arms to the ventral
nerve cord and form NMJs (Fig.
1). Unlike microspikes or myopodia, muscle arms are retained after
the formation of the NMJ and therefore provide a convenient model system for
the genetic analysis of muscle membrane extension.
Although several lines of evidence suggest that muscle arms are guided to
the nearest nerve cord (Hall and
Hedgecock, 1991; Hedgecock et
al., 1990), the nature of the guidance system is unknown. We and
others suggest that there are two phases of muscle arm development
(Dixon and Roy, 2005) (C. R.
Norris, I. A. Bazykina, E. M. Hedgecock and D. H. Hall, personal
communication). During embryogenesis, muscle arms probably arise through a
passive process that is initiated by juxtaposition between the myoblast and
the nascent motor axon. As the myoblast moves from the nerve cord, muscle
membrane remains connected to the nerve cord, resulting in an embryonic muscle
arm. By contrast, muscle arm extension during larval development is an active
process that requires regulators of the actin cytoskeleton, including
ADF/Cofilin (unc-60B) (Dixon and
Roy, 2005). It is unknown if microspikes, myopodia, and muscle
arms share common regulatory mechanisms.
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; Simmer et al.,
2003). We reasoned that some of these behavioral phenotypes may
result from defects in muscle membrane extension. Indeed, we found that
several integin and laminin components not only have EMEs, but also have
muscle arm extension defects (MADs) in which the larval muscle arms fail to
extend. Our results are consistent with a model whereby integrins and laminins
are needed for directed muscle arm extension to the nerve cords, while FGF
signaling provides a general mechanism to regulate membrane extension. |
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
resulting integrants were back-crossed to N2 (wild-type) three times. For
microscopy, worms were anesthetized in 2-10 mM Levamisole (Sigma) in M9
solution and mounted on a 2% agarose pad. All images were acquired with a
Leica DMRA2 compound microscope equipped with a Retiga 1300 digital camera (Q
imaging) using Open Lab (Improvision) software. Images were processed using
Volocity (Improvison) and Adobe Photoshop 7.0 (Adobe Systems). All muscle arm
counts were of the dorsal right-hand nerve cord. Unless otherwise indicated,
all counts were of muscles 9-21 and performed in the background of
RP247[trIs30], as described
(Dixon and Roy, 2005).
The RNAi screen for EMEs, scoring the EME phenotype and statistical analysis
Twenty-four RNAi by feeding vectors targeting known cell migration guidance
genes were constructed by inserting 0.8-1.5 kb fragments of genomic DNA
isolated by PCR from exon-rich regions of each gene into the EcoRI
site of the L4440 plasmid (Timmons and
Fire, 1998). These vectors were transformed into HT115 bacteria
(Timmons et al., 2001) using
standard protocols. RNAi by feeding vectors targeting let-60, ptp-2,
soc-2, egl-15 and 14 other RTK genes (Table S1 in the supplementary
material) and 847 Unc or Prz genes were from the Ahringer library
(Kamath et al., 2003). A
similar procedure was used for all RNAi-by-feeding experiments. L4-stage worms
(the Pos) were first plated onto 6 cm MYOB-agar plates lacking food
and allowed to run for 
0.5 hour to remove any contaminating OP50
bacteria. Then, for the screens of guidance and RTK genes, four worms were
re-plated into a 6 cm NGM-RNAi-inducing agar dish containing a single
RNAi-inducing bacterial clone. For the screen of 847 Unc or Prz genes two
Pos were re-plated into separate RNAi-seeded wells of a 12-well
dish. In both cases, after three days growth at 20°C, late L4 or young
adult F1 progeny were scored for the EME phenotype (+/–) at the
dissection microscope. Positive hits were re-tested and the EME phenotype was
quantified by counting the number of individual membrane processes extending
from the body wall muscles into the lateral hypodermal space. In each case,
the side of the animal was noted. Equal numbers of the left and right hand
sides were counted for each strain and averaged. All statistical analyses of
muscle arm counts and EMEs were performed using Student's t-test for
independent samples.
sem-5 and egl-15 rescue and cell specific RNAi
The ability of sem-5 to rescue the EME phenotype was tested by
injecting trIs10; sem-5(n1779) worms with a construct directing the
expression of sem-5 genomic DNA (sem-5g) either in BWMs
(myo3p::sem-5g) or pan-neuronally (F25B3.3p::sem-5g). The
presence of the rescuing arrays were followed in the BWMs and nervous system
with the co-injection reporters myo-3p::cfp and
F23B3.3::dsred2, respectively. Control strains contain the
co-injection markers plus pBluescript (Stratagene) DNA. The ability of
egl-15 to rescue the EME phenotype in muscle was tested by injecting
trIs10; egl-15(n484) worms with a construct directing the expression
of egl-15(5A) cDNA (courtesy of O. Hobert) from the myo-3
promoter. The presence of the rescuing arrays was followed with the
co-injection reporter myo-3p::cfp. A control line was also generated
using the same concentration of pBluescipt instead of
myo3p::egl-15(5A)cDNA. We also generated worms with an
Ex array directing the production of Mb::YFP in the BWM in the
background of oxIs14; egl-15(n484); otEx1270 and oxIs14;
egl-15(n484); otEx1269 (Bulow et al.,
2004). These otEx arrays drive expression of EGL-15A in
the hypodermis from the dpy-7 promoter and rescue the axon defects of
egl-15(n484) (Bulow et al.,
2004). As a negative control, cousins lacking the otEx
arrays were also counted. Before counting EMEs, we selected animals that
brightly fluoresced in the co-injection fluorescent channels to ensure that
animals carried the extra-chromosomal array in many of their cells.
Constructs directing the BWM-specific expression of dsRNA against gfp, unc-115, sem-5 and egl-15 gene products were generated by subcloning a coding-rich genomic fragment for each gene into the pPRSD102 plasmid, a derivative of pPD95.86 that contains an expanded MCS 3' to the myo-3 promoter, and isolating clones for gfp, unc-115, sem-5 and egl-15 fragments in both orientations with respect to the myo-3 promoter. To target mb::yfp mRNA for degradation, for example, two plasmids directing the production of RNA in opposite directions are co-injected along with the plasmid directing expression of a DsRed2 in BWMs to mark the array. All cell specific RNAi experiments were performed in a trIs10; sid-1(qt9) background. Multiple transgenic lines were isolated from each injection and from these lines, those worms that displayed robust expression of the co-injection marker in the body wall muscles were scored for EMEs.
Time-course data collection
Embryos from trIs10; egl-15(n484) and trIs10; sos-1(cs41)
worms were raised at either 20°C (for up-shift experiments) or 25°C
(for downshift experiments) for 12 hours, then harvested at room temperature
using the hypochlorite method and plated onto MYOB agar plates lacking food
(Lewis and Fleming, 1995). The
eggs were allowed to hatch overnight at either 20°C or 25°C without
food, causing them to arrest as L1 hatchlings. The next day, the synchronized
L1 population was divided and transferred to seven different plates and stored
at either 20°C or 25°C (the 0 hour time point). Individual plates were
transferred between 20°C and 25°C incubators when appropriate. For
example, in trIs10; egl-15(n484) up-shift experiments, one plate
containing L1s harvested and synchronized at 20°C was transferred to
25°C after 6 hours (the 6 hour time point), another plate transferred to
25°C after 12 hours (the 12-hour time point), etc. All strains were scored
for the EME phenotype upon reaching young adulthood.
let-756 rescue and mosaic analysis
let-756 rescue was tested using various promoters that drive
let-756 expression in different cell types. RP175 and RP176 are two
independent lines of the same genotype Ex[pPRRF186(let-756p::YFP);
(pPRRF187(let-756p::LET-756)]; let-756(s2887) unc-32(e189)III that were
used in a mosaic analysis to determine where let-756 expression was
required to rescue the lethality conferred by let-756(s2887). Both
RP175 and RP176 depend on the Ex array for viability, as no
non-fluorescent animals are ever observed in these two strains
(n>1000). We collected 47 RP175 and 95 RP176 animals that were
dramatically mosaic for the Ex array, being present in only a few
let-756-expressing cells. Of these, 10 RP175 animals had YFP
expression exclusively in the BWMs, five had YFP expression exclusively in the
pharynx and one had expression in only one CAN neuron. For RP176, 23 animals
had YFP expression exclusively in the BWMs, seven had YFP expression
exclusively in the pharynx and five had expression in only one or two CAN
neurons. We found no RP175 or RP176 animals with YFP expression in only the G1
and G2 glandular cells or only in the intestine.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) in all BWMs from a transgenic array called trIs10
(Fig. 1). Because BWMs of the
dorsal quadrants extend muscle arms to the dorsal cord, and the ventral BWMs
extend muscle arms to the ventral cord
(Dixon and Roy, 2005;
Hedgecock et al., 1990;
White et al., 1986), the left
and right lateral hypodermal ridges are devoid of BWM membrane extensions in
wild-type animals (Fig. 1). We
therefore expected that any EMEs projecting into the lateral space as a result
of disrupted plasma membrane extension would be readily visible in living RP1
animals. To test this, we examined the plasma membrane of BWMs in
unc-5 mutants. In unc-5 mutants, commissural motor axons
fail to extend to the dorsal cord and instead extend longitudinally along the
lateral and ventral hypodermal ridges
(Hedgecock et al., 1990).
Muscle arms from dorsal BWMs extend to these errant lateral and ventral motor
axons as a secondary consequence
(Hedgecock et al., 1990). As
expected, muscle arms that extend to the misguided axons are readily visible
in trIs10; unc-5(e53) animals
(Fig. 1), demonstrating that
trIs10 enables the visualization of BWM membrane in the lateral
hypodermal space.
We next investigated if genes required for the guidance of cell extensions
and migrations might also play a role in regulating BWM membrane extension.
Twenty-four genes required for guidance were targeted in an RNAi pilot screen
using RP1 worms (see Materials and methods and Table S1 in the supplementary
material). sem-5, which encodes a small adapter protein that is
orthologous to Grb2 and functions downstream of receptor tyrosine kinases
(Borland et al., 2001;
Clark et al., 1992;
Moghal and Sternberg, 2003;
Stern et al., 1993), was the
only RNAi target that resulted in EMEs
(Fig. 1). Two sem-5
hypomorphs, n1779 and n2019, both have significantly more
EMEs than the reporter strain alone (P<0.001,
Fig. 1C), confirming that
disruption of sem-5 results in the EME phenotype. We also observe
EMEs in sem-5(RNAi) animals in a reporter strain that expresses
DsRed2 throughout the BWMs, demonstrating that the EME phenotype is
independent of the Mb::YFP reporter (Fig.
1C). Furthermore, sem-5(RNAi) does not induce EMEs in all
cells, as the plasma membrane of the seam cells and DA and DB motoneurons in
animals treated with sem-5(RNAi) do not exhibit EMEs (n=100
animals, see Fig. S1 in the supplementary material). These results suggest
that sem-5 is required to prevent ectopic membrane extensions from
the BWMs.
The EMEs revealed by sem-5 hypomorphic activity often resemble the misguided muscle arms in mutants such as unc-5(e53). However, three lines of evidence suggest that EMEs are not a secondary consequence of misguided axons as in unc-5 mutants. First, the morphology and position of the DA, DB, DD and VD commissural motor axons in sem-5(RNAi)-treated RP1 animals is wild type (Fig. 1H,I). Second, disruption of sem-5 by RNAi in worms expressing a fluorescent reporter in all neurons (see Table S3 in the supplementary material) does not produce lateral axon guidance defects (n=10 animals). Third, EMEs do not extend to any particular lateral axon (data not shown), making it unlikely that EMEs extend into the lateral space because of altered lateral neuron fate. Together these results suggest that EMEs are not a secondary consequence of defects in neuronal development.
sem-5 expression in BWMs is both necessary and sufficient to prevent EMEs
To determine where sem-5 is required to regulate membrane
extension from the BWMs, we first examined the cells that express a YFP
reporter from 3.0 kb of promoter and enhancer sequences of
sem-5. Animals carrying this transgene express YFP in many cells
throughout development and in the adult, including the vulval precursor cells,
hypodermis, intestine, neurons and the BWMs (see Fig. S2 in the supplementary
material). To test if sem-5 expression in muscles is sufficient to
prevent the EME phenotype, we built a construct designed to express SEM-5
specifically in muscle cells from the myo-3 promoter
(myo-3p::SEM-5) (Okkema et al.,
1993). Three independent transgenic lines containing
myo-3p::SEM-5 were isolated in the background of the
sem-5(n1779) hypomorphic mutation. Each line displayed significantly
fewer EMEs than the negative control (P<0.001,
Table 1). By contrast,
pan-neuronal expression of SEM-5 does not rescue the EME phenotype
(Table 1). These results show
that sem-5 expression in muscle is sufficient to rescue the EME
phenotype.
|
) and
limit the RNAi effect to the muscles, we expressed the dsRNA in the background
of a sid-1(qt9)-null mutant (a kind gift from Craig Hunter). SID-1 is
required for dsRNA uptake into cells
(Feinberg and Hunter, 2003;
Winston et al., 2002).
Therefore the RNAi effect should be limited to the BWMs that express the
dsRNA. To test this approach, we injected trIs10; sid-1(qt9) animals
with three transgenes: two that direct the production of dsRNA against
gfp and a third that expresses nuclear-localized DsRed2 in muscle to
mark the presence of the extra-chromosomal (Ex) array that contains the
transgenes. We expected that YFP expression would be knocked down in muscles
that have the Ex array because gfp-coding sequence is 99% identical
to yfp, while neighboring muscles that do not have the Ex array
should maintain YFP expression. In trIs10; sid-1(qt9) animals mosaic
for the Ex array, 94% of DsRed2(+) BWMs were YFP(–) (n=143),
while 99% of DsRed2(–) BWMs were YFP(+) (n=79)
(Fig. 2A). Conversely, in
trIs10; sid-1(qt9) lines expressing dsRNA in BWMs targeting
unc-115, which is not expressed in muscles
(Lundquist et al., 1998) and
the sequence of which is unrelated to gfp, 100% of DsRed2(+) BWMs
were YFP(+) (n=201) (Fig.
2B). These results demonstrate that the RNAi effect can be
restricted to a cell of interest through the cell-specific expression of
dsRNA in the background of a sid-1 null mutation. To determine if sem-5 expression in the BWMs was necessary to prevent EMEs, we isolated four independent lines of trIs10; sid-1(qt9) animals expressing sem-5(dsRNA) specifically in BWMs and found that each had significantly more EMEs than negative controls (Fig. 2). Together, our results suggest that sem-5 expression in BWMs is both necessary and sufficient to negatively regulate ectopic membrane extension from the BWMs.
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;
Moghal and Sternberg, 2003;
Stern et al., 1993). To
determine if sem-5 regulates muscle membrane protrusions downstream
of an RTK, we screened 14 C. elegans RTKs genes for the EME phenotype
using RNAi (see Table S1 in the supplementary material). egl-15,
which encodes the lone FGF receptor (FGFR) in C. elegans
(DeVore et al., 1995), was the
only RTK that resulted in the EME phenotype
(Table 1). Three
egl-15 hypomorphic alleles that we examined also have EMEs
(Fig. 3A,B,
Table 1). Although BWM spatial
expression of egl-15 has not yet been reported, both SAGE analysis
and Affymetrix microarray analysis of cultured C. elegans myoblasts
reveals low, but significant, expression of egl-15 mRNAs (Don Moerman
and David Miller, III, personal communication). Knocking down egl-15
activity specifically in BWMs using the same RNAi approach outlined above for
sem-5 results in significantly more EMEs than control strains
(Fig. 2D). Moreover, a
construct directing the expression of an egl-15 cDNA from the
myo-3 promoter in the BWM rescues the EME phenotype of trIs10;
egl-15(n484) worms (Table
1). Conversely, expression of egl-15 in the hypodermis
does not rescue the EME phenotype (Table
1, lines 32-35). These results suggest that FGFR activity within
the BWMs is required to prevent the EME phenotype.
We next tested if other known FGF pathway genes play a role in regulating
membrane extension from the BWMs. There are two putative FGF ligands upstream
of egl-15 encoded by egl-17
(Burdine et al., 1997) and
let-756 (Roubin et al.,
1999). The number of EMEs in egl-17(n1377) null animals
did not differ from negative controls (P>0.05). By contrast,
let-756(s2613) hypomorphs had significant numbers of EMEs
(P<0.001) (Fig. 3C,
Table 1), suggesting that
LET-756 is the ligand necessary to prevent EME formation. Numerous genes
function downstream of egl-15, including the adapter proteins
sem-5(Grb2), soc-1(Gab1) and soc-2(Shoc-2/Sur-8),
the non-receptor tyrosine phosphatase ptp-2(Shp2), the Ras guanine
nucleotide exchange factor sos-1 (Son of Sevenless), and the
Ras-family GTPase let-60 (Ras)
(Borland et al., 2001). We
determined that soc-2(RNAi), ptp-2(RNAi) and let-60(RNAi)
each induced EMEs (Fig. 3D,
Table 1). EMEs were also
observed in strains containing soc-1, sos-1 and let-60
hypomorphic alleles (Table 1).
Together, these data suggest that FGF signaling negatively regulates ectopic
membrane extensions from the BWMs.
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) and sos-1(cs41)
(Rocheleau et al., 2002) (see
Fig. 4 and Materials and
methods for details). Both egl-15(n484) and sos-1(cs41)
worms shifted to 25°C before the third larval stage had more EMEs than the
negative control animals kept at 20°C, while those shifted after the third
larval stage did not (Fig.
4A,B). Conversely, mutant L1 hatchlings raised at 25°C and
then immediately shifted to 20°C for the remainder of larval development
show no more EMEs than animals raised continuously at 20°C
(Fig. 4C,D), indicating that
the FGF pathway is not required during embryogenesis to regulate EMEs. The
number of EMEs in animals left in the non-permissive temperature significantly
increased at each successive time point until the middle of the third larval
stage at the 24 hour time point, when the number of EMEs was not different
than animals raised constitutively at 25°C. These results show that FGFR
signaling is required specifically during early to mid larval development to
prevent EME formation. Larval muscle arm extension occurs exclusively during
this same period (Dixon and Roy,
2005), suggesting that the FGF pathway functions to prevent
widespread ectopic extension of the muscle membrane during this crucial
period.
To investigate the effects of upregulation of the FGF pathway on membrane
extension, we examined BWM plasma membrane in a clr-1
loss-of-function mutant. clr-1 encodes a BWM-expressed receptor
tyrosine phosphatase that negatively regulates EGL-15 activity
(Kokel et al., 1998). The
clr-1(e1745) temperature-sensitive allele can suppress the EMEs
induced by egl-15 and sem-5 hypomorphic activity
(Fig. 3E,F,
Table 1), consistent with the
role of CLR-1 in antagonizing ELG-15-mediated signaling. Similarly, the
let-60(n1046) hypermorph is able to partially suppress the EME
phenotype induced by sem-5(RNAi) and egl-15(RNAi)
(Fig. 3G,H;
Table 1). These data are
consistent with previous work showing that let-60 functions in the
FGF pathway, but does not mediate all aspects of EGL-15 function
(DeVore et al., 1995;
Sundaram et al., 1996).
Interestingly, clr-1(e1745) mutants have a dramatic reduction in
the number of muscle arms compared with controls
(Fig. 3I,J,
Table 2), suggesting that
larval muscle arms fail to extend in these mutants
(Dixon and Roy, 2005). The MAD
phenotype of clr-1 mutants is partially suppressed by
sem-5(RNAi) and completely suppressed in an egl-15(n1783)
background (Fig. 3L,M,
Table 2). These results suggest
that the level of EGL-15 activity is a key determinant of the ability of the
BWM to extend plasma membrane: decreased EGL-15 activity results in ectopic
membrane extensions, while increased EGL-15 activity abrogates larval muscle
arm extension.
|
; Popovici et al.,
2004) (see Fig. S2 in the supplementary material). Although the
same cells expressed a LET-756::YFP fusion protein driven from the
let-756p, we also observed nuclear localization of LET-756::YFP,
along with pericellular expression around the BWMs that is very similar to
that of CLR-1 (Kokel et al.,
1998) (Fig.
5A-D). We rescued the lethal phenotype of let-756(s2887) null mutants with an extra-chromosomal array that contained both the let-756p::YFP reporter and a second construct that used the same let-756p to drive the expression of let-756 genomic coding sequence. Using the let-756p::YFP reporter to mark the cells that expressed LET-756 from the rescuing array, we determined that LET-756 expression in a single CAN neuron, a single BWM or cells in the pharynx was sufficient to rescue the lethality of let-756(s2887) nulls. Importantly, we could find no let-756(s2887) homozygotes that did not express YFP (n>1000), indicating that physiologically relevant LET-756 is not expressed in cells that do not also express YFP from the array. Moreover, when only a small number of BWMs or pharynx cells had the rescuing array in let-756(s2887) homozygotes, the animal had a pronounced bulge near the site of YFP expression, suggesting local rescue of the scrawny phenotype (Fig. 5E,F). Together, our observations demonstrate that transgenic expression of let-756 from any one of a number of distinct cell types is sufficient to rescue the lethal phenotype of let-756 nulls.
As let-756 expression from distinct cell types rescued the lethal phenotype of let-756 nulls, we tested if the same was true for the EME phenotype. We observed complete rescue of both lethality and EMEs in let-756(s2887) nulls expressing LET-756 from the endogenous let-756p, in neurons from the pan-neuronal F25B3.3 promoter or in the hypodermis from the dpy-7 promoter (Fig. 5G). This suggests that LET-756 functions non-autonomously to negatively regulate EMEs from the body wall muscles.
Disruptions of integrin and laminin result in both EMEs and MADs
Postsynaptic termini of the neuromuscular junction reside on the distal
ends of muscle arms (Dixon and Roy,
2005; Stretton,
1976; Sulston and Horvitz,
1977; White et al.,
1986). We reasoned that disruption of muscle arm extension might
therefore result in locomotory defects. In an effort to identify additional
genes that regulate muscle membrane extension, we screened 847 genes that
result in an uncoordinated or paralyzed phenotype when targeted by RNAi
(Kamath and Ahringer, 2003;
Simmer et al., 2003), 23 of
which resulted in a strong EME or MAD phenotype (M.A., R.F., S.J.D. and
P.J.R., unpublished). One of these genes, lam-1, encodes the only
laminin ß-subunit in C. elegans
(Huang et al., 2003;
Hutter et al., 2000) and
results in significant EMEs when disrupted by RNAi or mutation
(Fig. 6,
Table 3). pat-4, which
encodes an integrin-linked kinase ortholog
(Mackinnon et al., 2002), and
pat-6, which is required for the assembly of integrin complexes
(Lin et al., 2003), were also
identified in our screen (Table
3). These results prompted us to investigate other integrin and
laminin genes (Cox and Hardin,
2004; Hutter et al.,
2000). epi-1 (
B-laminin),
lam-1 (ß-laminin), lam-2 (
-laminin) and
pat-2 (-integrin) each induced EMEs when targeted by RNAi and
had significant reductions in the number of muscle arms reaching the nerve
cord in either RNAi or hypomorphic mutant backgrounds
(Fig. 6,
Table 2). By contrast, other
cell-adhesion genes that we examined by RNAi or mutations, such as all 13
predicted cadherin genes (Cox et al.,
2004), conferred no EMEs (not shown). Similarly, disruption of
ina-1 (-integrin) by RNAi or mutation did not result in EMEs
(Table 2). This was a
surprising result, as the INA-1/PAT-3 integrin heterodimer probably binds
laminin, while the PAT-2/PAT-3 complex probably binds UNC-52/perlecan
(Baum and Garriga, 1997;
Rogalski et al., 1993).
However, this prompted us to examine perlecan mutants. We found that
unc-52(e998) mutants phenocopy the MAD and EME phenotypes of
pat-2(RNAi) (Fig. 6,
Table 2). These data suggest
that the PAT-2/PAT-3 integrin complex functions with laminins and perlecan to
regulate muscle membrane extension.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), providing additional evidence that the LET-756 FGF pathway
is required during this period to inhibit EMEs. Although FGFR signaling acts
in muscles to regulate membrane extension, let-756 can be expressed
from a variety of spatial locations to prevent the formation of the ectopic
membrane extensions without compromising muscle arm extension. This argues
against LET-756 acting as either an attractant or repellant of muscle membrane
extension. Instead, we speculate that the interaction of LET-756 with EGL-15
acts as a switch to negatively regulate membrane extension by unknown
molecular mechanisms.
Unlike the FGF pathway, we found that disruptions in laminin, integrin and
perlecan result in both ectopic membrane extensions and fewer muscle arms
reaching the nerve cord. These phenotypes are consistent with wayward muscle
arm extensions that fail to reach their target. We speculate that the
laminins, integrins and perlecan, which together constitute a significant part
of the extracellular matrix and muscle adhesion complex
(Cox and Hardin, 2004,
Hutter et al., 2000), could be
required to regulate the spatial distribution of factors that guide muscle
arms to the nerve cord. Alternatively, disruption of laminin, integrin and
perlecan may interfere with proper localization of receptors on the plasma
membrane of muscles or with adhesion of the muscle membrane extensions to the
substratum (Huang et al.,
2003). Under any model, the ectopic membrane extensions resulting
from compromised laminin and integrin function depend on FGF pathway activity
as increased FGFR activity suppresses their extension.
|
;
Burdine et al., 1997). The
egl-15 gene encodes two isoforms by splicing between two alternative
exons called 5A and 5B (Goodman et al.,
2003). Null mutations that are specific to the 5A exon, including
egl-15(n484), reveal that only EGL-15(5A) is necessary for the
guidance of the sex myoblasts and is therefore likely to be the only isoform
on the sex myoblasts required to bind EGL-17
(Goodman et al., 2003).
Although egl-15(n484) and let-756(s2613) mutations result in
EMEs from the BWMs, the egl-17(n1377) null allele does not. Our
observations therefore suggest that EGL-15(5A) is capable of receiving either
EGL-17 or LET-756 ligands, depending on the cellular context. This conclusion
is further supported by the fact that expression of LET-756 under the control
of egl-17 regulatory elements can partially rescue the sex-myoblast
migration defects (Goodman et al.,
2003).
The second well-established role for FGFR signaling is in fluid
homeostasis. Hyperactivity of EGL-15 results in fluid filled worms, called a
clear or Clr phenotype (Kokel et al.,
1998). By contrast, hypomorphic let-756 or
egl-15 alleles that do not specifically disrupt EGL-15A result in a
scrawny phenotype that is consistent with the worms lacking sufficient fluid
(DeVore et al., 1995;
Roubin et al., 1999). The null
phenotype of let-756 or egl-15 is early larval lethality,
which probably results from improper fluid regulation
(Birnbaum et al., 2005;
Huang and Stern, 2005). Recent
work has demonstrated that EGL-15 activity in the hypodermis is essential for
fluid regulation and that LET-756 probably signals to EGL-15 in the hypodermis
from the BWMs (Huang and Stern,
2004). These results are seemingly contradictory to our
observations that LET-756 expression from a transgenic array can rescue the
essential let-756 function from any number of cells, including a
single BWM, a single CAN neuron or a limited number of cells in the pharynx.
However, this difference can be reconciled by noting that the specific
requirement of LET-756 in BWMs was determined by Huang and Stern
(Huang and Stern, 2004) using
a free chromosomal duplication, while we used a transgenic array that
typically results in transgene overexpression
(Mello et al., 1991). We
therefore suggest that overexpression of LET-756 from few cells of any type
might be sufficient to regulate fluid balance and that the ligand can travel
some distance within the animal to trigger EGL-15 activity.
A third role for EGL-15 is in axon extension, guidance and the positional
maintenance of two interneuron axons along the ventral midline
(Bulow et al., 2004). We
believe that EMEs are not a secondary consequence of compromising these roles
of EGL-15 for many reasons. First, the egl-15(n484) allele has no
axonal guidance defects (Bulow et al.,
2004), but does result in severe EMEs. Second, the cellular focus
of EGL-15 in axon extension and guidance is in the hypodermis
(Bulow et al., 2004), whereas
we have demonstrated that egl-15 and sem-5 are both
autonomously required within the BWMs to antagonize EMEs. Third, EGL-15 is
required embryonically for axon extension and guidance
(Bulow et al., 2004), whereas
our temperature-shift time-course experiments using temperature-sensitive
alleles demonstrated that egl-15 and sos-1 are dispensable
during embryonic development to prevent EMEs. Fourth, the motor axon extension
and guidance defects of egl-15 nulls are relatively minor, being
restricted to a single anterior commissure
(Bulow et al., 2004), whereas
we observe EMEs all along the entire length of the animal. Fifth, the role of
EGL-15 in the maintenance of axon position is restricted to two ventral cord
interneurons and does not require the intracellular domains of EGL-15
(Bulow et al., 2004). These
observations are inconsistent with EMEs being induced by defects in the
maintenance of axon position because the egl-15(n1783) mutation that
specifically disrupts the EGL-15 kinase domain
(Goodman et al., 2003) results
in EMEs, along with other intracellular transducers of the EGL-15 signal,
including sem-5, sos-1 and let-60. Last, we did not observe
EMEs projecting to any particular axon in the lateral hypodermal ridge in FGFR
mutants. We therefore conclude that the aberrant membrane projections that
result from compromised FGFR signaling are not a secondary consequence of
defects in axon extension, guidance or positional maintenance.
Finally, an FGF pathway is required for acetylcholine receptor (AChR)
expression at the neuromuscular junction in C. elegans
(Gottschalk et al., 2005).
egl-15 acts in parallel with cam-1, which encodes a
ROR-family receptor tyrosine kinase ortholog, to regulate the presence of
AChRs at the NMJ, without altering the transcription levels of AChR subunits
(Gottschalk et al., 2005). We
have examined several mutants involved in neurotransmission at the
neuromuscular junction, including cam-1/ROR
(Francis et al., 2005),
unc-18/UNC-18 (Weimer et al.,
2003), cha-1/choline acetyltransferase
(Alfonso et al., 1994),
unc-29/nicotinic AChR subunit
(Fleming et al., 1997) and
unc-25/glutamic acid decarboxylase
(Jin et al., 1999), none of
which have muscle membrane extension defects (S.J.D. and P.J.R., unpublished).
This suggests that neurotransmission does not regulate muscle membrane
extension. However, the work of Gottschalk and colleagues raises the exciting
possibility that the FGF pathway may regulate the localization or membrane
insertion of other receptors that regulate BWM membrane extension.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/7/1263/DC1
1 Collaborative Program in Developmental Biology, University of Toronto,
Toronto, ON, M5S 1A8, Canada. 
|
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