Yale University School of Medicine, Department of Genetics, I-354 SHM, PO Box 208005, New Haven, CT 06520-8005, USA
* Author for correspondence (e-mail: michael.stern{at}yale.edu)
Accepted 31 August 2004
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SUMMARY |
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Key words: FGF receptor, EGL-15, PI3 kinase, Signal transduction, Muscle differentiation, M lineage, Myoblast, SM, Sex muscles
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Introduction |
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C. elegans is an excellent model organism in which to study the
complex interplay of signaling pathways that regulate myogenesis.
Post-embryonic muscles are derived from a single blast cell, the M mesoblast
(Sulston and Horvitz, 1977).
In hermaphrodites, M undergoes several rounds of cell division in the first
larval stage to give rise to 14 body wall muscles (BWMs), two dorsally
positioned coelomocytes and two sex myoblasts (SMs). The SMs, which are born
in the posterior body region of the animal, migrate anteriorly during the
second larval stage to flank the precise center of the developing gonad, the
site of the future vulva and uterus. Midway through the third larval stage,
the two SMs undergo three rounds of cell division to produce a total of 16
cells. During the fourth larval stage, these cells differentiate into the
mature egg-laying muscles, alternatively known as the sex muscles. The sex
muscles comprise two types of uterine muscles, um1s and um2s, and two types of
vulval muscles, vm1s and vm2s. These cells assume highly reproducible
morphologies and positions (Sulston and
Horvitz, 1977
), and they express molecular markers characteristic
of differentiated muscle cells, including F-actin and other components of the
contractile machinery, as well as other markers more specific to the sex
muscles (Harfe et al., 1998a
;
Moerman and Fire, 1997
).
FGF signaling plays an important role in the development of the
hermaphrodite sex muscles by helping to guide the migrating SMs. The FGF-like
ligand EGL-17 serves to attract the SMs to their precise final positions
(Burdine et al., 1998). The
receptor for this chemoattractant is encoded by egl-15, the sole FGF
receptor gene in C. elegans
(DeVore et al., 1995
).
egl-15 is alternatively spliced to generate two isoforms termed
EGL-15(5A) and EGL-15(5B). The 5A isoform is required for SM chemoattraction,
while the 5B isoform is required to mediate an essential function of EGL-15
(Goodman et al., 2003
).
Mutations that perturb SM chemoattraction, either by eliminating the EGL-17
FGF or the EGL-15(5A) isoform, cause the SMs to be severely posteriorly
displaced (Burdine et al.,
1998
; Goodman et al.,
2003
). Even when severely mispositioned, the SMs still proliferate
normally and generate cells that appear to differentiate properly (I.E.S.,
unpublished). Owing to their abnormal positions, however, these muscle cells
are unable to attach and function properly, resulting in an egg-laying
defective (Egl) phenotype.
Hyperactivating EGL-15 has provided an important approach for understanding
the role of EGL-15 in C. elegans development and physiology
(Kokel et al., 1998;
Selfors et al., 1998
). EGL-15
can be hyperactivated by mutational inactivation of clr-1, a gene
encoding a receptor tyrosine phosphatase that negatively regulates EGL-15
activity (Kokel et al., 1998
).
clr-1 mutants accumulate fluid within the pseudocoelomic cavity,
resulting in a translucent or clear (Clr) appearance. A similar Clr phenotype
results from directly hyperactivating EGL-15 via transgenic expression of the
egl-15(neu*) construct
(Kokel et al., 1998
). This
construct replaces the transmembrane domain of EGL-15 with that of oncogenic
Neu, presumably causing constitutive dimerization of EGL-15. A
temperature-sensitive mutation in clr-1 has been extremely useful in
helping to define a canonical FGF signaling pathway in C. elegans
(Kokel et al., 1998
;
Schutzman et al., 2001
;
Selfors et al., 1998
) as well
as other functions of this pathway
(Szewczyk and Jacobson, 2003
).
The usefulness of transgenic egl-15(neu*), however, has
been limited, as the constitutive Clr phenotype that it confers prohibits the
establishment of heritable transgenic lines. By suppressing the Clr phenotype
conferred by transgenic egl-15(neu*), we have been able to
assess other effects of hyperactivated FGF signaling on C. elegans
development. Using this approach, we have discovered that EGL-15
hyperactivation can block sex muscle differentiation. This effect is
consistent with the ability of FGF to inhibit vertebrate myoblast
differentiation and can be used to identify signaling components that
influence myogenesis in vivo.
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Materials and methods |
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Constructs and germline transformation
Transgenic arrays were generated using standard germline transformation
techniques (Mello et al.,
1991). Plasmids generated using PCR were confirmed by sequence
analysis. Integrated strains were backcrossed at least four times prior to
analysis.
Control strains
ccIs4251 is an integrated array of the pSAK2 plasmid
(Fire et al., 1998), which
expresses GFP under the control of the Pmyo-3 myosin heavy
chain promoter. ccIs4251 is used as a control integrated array for
the pSAK2 germline transformation marker. ayIs29 was
isolated as a spontaneous integrant of an array carrying the egl-15
genomic rescuing fragment NH#112 (5 ng/µl)
(DeVore et al., 1995
), the
Pmyo-2::GFP transformation marker pJKL449.1 (5 ng/µl)
and pGEM-5Z (70 ng/µl).
egl-15(neu*)
Germline transformants of NH#420 (20 ng/µl)
(Kokel et al., 1998) were
isolated in a soc-2 background using pSAK2 (5 ng/µl) as a
co-transformation marker. Two independent genomic integrants, ayIs15
and ayIs16, were isolated using a standard gamma irradiation protocol
(1251.3 rads) (Mello and Fire,
1995
). egl-15(neu*) animals display multiple
pleiotropies. Some animals display a mild uncoordinated phenotype that is most
prominent in the adult, and rare animals display a multi-vulva phenotype.
Isoform specific neu* constructs
The egl-15(5B*) construct (NH#1033) was generated by
replacing the extracellular coding sequence of NH#420 with the corresponding
sequence containing a nonsense mutation in exon 5A from NH#872
(Goodman et al., 2003). The
egl-15(5A*) construct (NH#1034) was generated similarly,
using NH#873 as a source of a nonsense mutation at the first codon of exon 5B
(Goodman et al., 2003
).
Germline transformants of NH#1033 (20 ng/µl) were isolated in a
soc-2 background using pSAK2 (5 ng/µl) as the co-transformation
marker. Germline transformants of NH#1034 (20 ng/µl) were isolated in an N2
background using pSAK2 (5 ng/µl) as the co-transformation marker and
crossed into a soc-2(n1774) background. Two independent integrated
NH#1033 arrays, ayIs17 and ayIs18, were isolated using a
standard gamma irradiation protocol (1247 rads)
(Mello and Fire, 1995
). Two
independent integrated NH#1034 arrays, ayIs21 and ayIs27,
were generated using 50 µg/ml TMP and 350 uJ(x100) UV light (Scott
Clark, personal communication).
The effect of extrachromosomal egl-15(5A*) arrays appears to differ somewhat from that of integrated arrays. Both types of arrays behave similarly with regard to their effects on the Clr phenotype, failing to confer a Clr phenotype in a soc-2(+) background. In contrast to the strongly penetrant Egl phenotype conferred by integrated egl-15(5A*) arrays, several extrachromosomal arrays expressing egl-15(5A*) display only a partially penetrant Egl phenotype. The behavior of the integrated egl-15(5A*) arrays appears to be bona fide, as multiple integrated arrays display the identical 100% penetrant Egl phenotype, including those isolated from different extrachromosomal arrays that were generated in independent experiments. The reduced penetrance of the Egl phenotype in animals bearing extrachromosomal arrays may result from the mosaicism of the extrachromosomal arrays as well as in part to eliminating the EGL-15(5B*) contribution.
Tissue-specific expression of egl-15(neu*)
Phlh-1 (Krause et
al., 1990), Punc-54
(Okkema et al., 1993
),
Punc-14 (Ogura et al.,
1997
) and Paex-3
(Iwasaki et al., 1997
) were
used to express EGL-15(neu*) in a variety of tissue types.
Construction of the Punc-54 (NH#1181),
Punc-14 (NH#1094) and Paex-3 (NH#1190)
plasmids have been described by Huang and Stern
(Huang and Stern, 2004
). The
Phlh-1 constructs were generated by PCR amplification of
genomic DNA and include the region from 2960 to 1 bp 5' to
the start ATG. The PCR product was cloned upstream of the egl-15
genomic sequence (NH#150) to generate Phlh-1::egl-15
(NH#1201), upstream of egl-15(neu*) (NH#526) to generate
Phlh-1::egl-15(neu*) (NH#1198), and upstream of
the kinase-defective egl-15(neu*K672A) (NH#527) to
generate Phlh-1::egl-15(neu*K-A) (NH#1199).
Germline transformants expressing each construct (20 ng/µl), except Punc-14::egl-15(neu*), were generated in an N2 background using pJKL449.1 (5 ng/µl) as a co-transformation marker. Punc-14::egl-15(neu*) expressed in an N2 background results in a Clr phenotype that precludes the isolation of stably transmitted lines. Therefore, Punc-14::egl-15(neu*) lines were established in a soc-2 background. Heritable lines expressing Phlh-1::egl-15(neu*) were established by selecting GFP+ mosaic animals that were normal in appearance. Transgenic animals that maintained the array in the germline segregated some progeny that display a severe lumpy-dumpy phenotype.
Serotonin response assay
The serotonin response assay followed standard procedures
(Schafer and Kenyon, 1995),
and was modified by culturing young adult animals on unseeded 2% agar plates
with 5 mM 5-hydroxytryptamine creatinine sulfate monohydrate (ICN Biomedicals)
for 1 hour. Animals were subsequently removed from serotonin plates, and the
number of eggs laid/animal was scored. At least 12 animals were assayed per
data point, and the mean and standard deviation were calculated.
Egg-laying index
Animals were synchronized by L1 arrest and then fed for 44 hours. At the
late L4 Christmas tree stage, individual animals were cloned and allowed to
lay eggs, which were counted as larvae 48 hours later (number of laid eggs).
After 24 hours, gravid adults were then removed, lysed in 20% bleach and the
eggs remaining in the uterus were counted (number of unlaid eggs). The
egg-laying index= (number of laid eggs)/(number of laid + number of unlaid
eggs).
Rhodamine-phalloidin staining
Animals were synchronized by L1 arrest followed by feeding for 60 hours.
Young adults were then harvested by washing three times in M9 and fixed in
3.7% formaldehyde in 0.1M Na2HPO4 for 3 hours with
gentle rocking. Animals are washed three times in PBS, dehydrated in ice-cold
acetone for 2 minutes, and washed three times in PBS. Packed worms (30 µl)
were then stained with rhodamine-conjugated phalloidin (Molecular Probes) at
1:50 dilution for 3 hours at 20°C in the dark. Worms were repeatedly
washed for 30 minutes in PBS. Both Pmyo-3::GFP expression
and rhodamine-phalloidin staining can be used to monitor the differentiation,
morphology and position of the vulval muscles
(Moerman and Fire, 1997);
contractile fibers in the uterine muscles appear more loosely organized and
cannot be reproducibly monitored by either of these two methods.
Anti-EGL-15 staining
Young adults were harvested for each strain and stained following the
standard protocol of Finney and Ruvkun
(Finney and Ruvkun, 1990). Two
affinity-purified anti-EGL-15 primary antibodies (Pop, Crackle) were used at a
1:10 dilution; Alexa Fluor 546-conjugated anti-rabbit antibody (Molecular
Probes) was used as a secondary antibody at a 1:250 dilution. EGL-15
antibodies detect the sixteen undifferentiated cells of the SM lineage, the
differentiated vm1s, several neurons in the head and the hypodermis during
L1-L3 stages. A more detailed analysis will be described elsewhere.
Identification of M lineage defects
A combination of DIC microscopic examination and the integrated
Ptwist::GFP marker, ayIs6, was used to assess the
development of the early M lineage, the sex myoblasts (SMs) and the dorsal
coelomocytes (dccs). Ptwist::GFP is expressed in all
undifferentiated cells of the M lineage
(Harfe et al., 1998b) and was
used to score the positions of undifferentiated cells in the early M lineage
(L1 stage). Coelomocytes were scored by DIC. The final positions of the SMs
were scored as previously described
(Thomas et al., 1990
). SM
distributions are represented as box-and-whisker plots
(Moore and McCabe, 1993
) as
described by Goodman et al. (Goodman et
al., 2003
).
Photomicroscopy
All micrographs were collected using a Hamamatsu C4742-95 digital camera
mounted on a Zeiss Axioskop microscope that contains an internal 0.63x
reduction lens. Images were collected as a z-series of 0.5 µm
sections, merged into a single composite image and false colored using OpenLab
software. Images were then cropped and scaled using Adobe Photoshop.
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Results |
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Abnormalities in the sex muscles could also account for a
serotonin-resistant egg-laying defect. The distinctive architecture of the
vulval and body wall muscles can be identified in wild-type animals using
rhodamine-phalloidin, which binds to the highly organized filamentous actin
cables of C. elegans muscles (Fig.
3A,B) (Moerman and Fire,
1997). These cell types can also be assessed based on their
expression of the Pmyo-3::GFP transformation marker.
myo-3 encodes one of two myosin heavy chain isoforms that are
expressed in all non-pharyngeal muscles, including the body wall muscles and
the vulval muscles (Ardizzi and Epstein,
1987
; Dibb et al.,
1989
; Miller et al.,
1983
). In contrast to wild-type animals, no sex muscle staining is
observed in soc-2; ayIs15 animals
(Fig. 3C;
Table 2) despite a normal
pattern of rhodamine-phalloidin staining for the body wall musculature.
Consistent with this observation, soc-2; ayIs15 animals express
Pmyo-3::GFP at normal levels in the body wall muscles, but
lack the strong Pmyo-3::GFP expression normally observed
in the vulval muscles (Fig. 3C;
Table 2). Thus, soc-2;
ayIs15 animals appear to be unable to lay eggs because of the lack of a
normal set of sex muscles.
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Consistent with the normal functions of the isoforms, EGL-15(5B*) was found to be primarily responsible for the Clr phenotype of egl-15(neu*), while EGL-15(5A*) showed a stronger effect on the egg-laying muscles. EGL-15(5B*) arrays confer a robust Clr phenotype in a soc-2(+) background, similar to that observed for intact egl-15(neu*) (Table 1). Similar effects were seen using either extrachromosomal or integrated 5B* arrays (ayIs17 and ayIs18). When these arrays were expressed in a soc-2 background, the Clr phenotype was suppressed, as in ayIs15 (Table 1). These animals were also significantly Egl, although distinguishable from the soc-2; ayIs15 Egl phenotype; 5B* animals retain some egg-laying capability (Fig. 2B) and have differentiated sex muscles, as determined by rhodamine-phalloidin staining and Pmyo-3::GFP expression (Fig. 3D; Table 2).
By contrast, expression of EGL-15(5A*) alone can result in animals that completely lack differentiated sex muscles, but fail to develop a Clr phenotype. Wild-type animals expressing egl-15(5A*) from extrachromosomal arrays appear non-Clr, as do two independently isolated integrated lines (ayIs21 and ayIs27; Table 1). Both integrated arrays confer a completely penetrant Egl phenotype identical to that of soc-2; ayIs15 (Fig. 2B, Tables 1, 2). Similar to soc-2; ayIs15 animals, ayIs21 and soc-2; ayIs27 animals fail to generate sex muscles that express Pmyo-3::GFP or stain with rhodamine-phalloidin (Fig. 3E; data not shown). This constellation of phenotypes is consistent with the 5A isoform playing an important role specifically in the M lineage and with the ability of the 5A* isoform to interfere with the development of differentiated sex muscles.
egl-15(neu*) inhibits sex muscle differentiation
FGF signaling is known to inhibit muscle differentiation during vertebrate
myogenesis (Itoh et al.,
1996), suggesting that egl-15(neu*) might
block the differentiation of the hermaphrodite sex muscles. To test whether
the sex muscles were failing to differentiate, we used anti-EGL-15 antibodies
to visualize undifferentiated SM descendants. These antibodies are only
capable of detecting EGL-15 when expressed from transgenic arrays, which is
probably due to the low-level expression of the endogenous receptor.
Wild-type egl-15 transgenes express EGL-15 in the SMs and their descendants in the L3 and L4 larval stages. In the adult hermaphrodite, EGL-15 expression in the SM lineage becomes restricted to the type 1 vulval muscles, which display a characteristic morphology and position flanking the vulva (Fig. 4A,B). By contrast, adult animals expressing EGL-15(neu*) have many more cells that express EGL-15. The number and position of these cells is consistent with the number and position of the SM descendants observed by Nomarski microscopy (Fig. 6B; data not shown). The aberrant positions of these cells probably result from a combination of the prior defects in cell division polarity and SM migration (as described below). These cells have rounded cell bodies with projections that extend anteriorly and posteriorly (Fig. 4C,D), a morphology very distinct from that of differentiated sex muscle cells. The undifferentiated morphology of these cells, their failure to restrict egl-15 expression, the weak expression of Pmyo-3::GFP, and the absence of filamentous actin all indicate that these cells fail to differentiate correctly, resulting in a lack of differentiated sex muscles.
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Hyperactivation of EGL-15 results in multiple M lineage defects
To better understand the developmental defects that result in the abnormal
staining pattern observed in soc-2; egl-15(neu*) animals
(Fig. 4C), we examined the
sequence of developmental events from the divisions of the post-embryonic M
mesoblast to the differentiation of the sex muscles. Multiple aspects of sex
muscle development are affected by hyperactivation of EGL-15, including the
polarity of the cell divisions in the early M lineage, cell fate determination
events and SM positioning.
The earliest M lineage defect detected in soc-2; ayIs15 animals
was an abnormal number of M-derived cells in the four muscle quadrants. Cells
in the M lineage were identified using a Ptwist::GFP
reporter (ayIs6 [Ptwist::GFP])
(Harfe et al., 1998b). In
wild-type animals, the first division of M is oriented along the dorsoventral
axis. Each M daughter cell (M.x) then divides transversely (left-right). The
orientations of these first two divisions position a single M-derived muscle
progenitor cell in each of the four-muscle quadrants. These cells subsequently
divide along the anteroposterior axis to generate a total of 14 body wall
muscles, two coelomocytes and two sex myoblasts
(Fig. 6A)
(Sulston and Horvitz, 1977
).
Because of the tight linkage between the ayIs6 and ayIs15
transgenes, the second egl-15(neu*) integrated array,
ayIs16, was used for this analysis. In soc-2; ayIs16
animals, the polarities of the early M-lineage divisions are often abnormal.
This can be scored most easily for divisions of M.x or M.xx, where division
polarity defects result in muscle quadrants lacking M-derived descendants.
When soc-2; ayIs16 animals are scored in this manner, the majority of
soc-2; ayIs16 animals display muscle quadrants lacking M-derived
cells (Fig. 6B). A similar
defect is seen in 5A* animals and, to a reduced extent, in
5B* animals. Despite the division polarity defect, the
number of M descendants generated during the first larval stage still appears
to be normal (data not shown).
Additional defects within the M lineage were also readily apparent in animals with hyperactive EGL-15 signaling. The two types of non-body muscle M descendants, the SMs and the dorsal coelomocytes, are sufficiently distinctive in their appearance and in their expression of the ayIs6 marker that their presence can be scored independent of earlier division polarity defects. A dramatic absence of the distinctive M-derived coelomocytes was observed in animals bearing an egl-15(neu*) array (Fig. 6B), suggesting that hyperactivated EGL-15 signaling could interfere with their normal fate determination or differentiation. In addition, the normal precise positioning of the SMs was disrupted by EGL-15 hyperactivation. Both of these defects were apparent regardless of whether or not these cells were generated in their proper quadrants. Similar effects were also observed in 5A* animals and, to a reduced extent, in 5B* animals. The effects of egl-15(neu*) on multiple events that occur in the M lineage highlight the crucial role that FGF signaling can play during development.
Activation of PI3 kinase signaling can suppress the muscle differentiation defect of egl-15(neu*)
Biochemical and cell culture analyses have shown that insulin, which acts
through a phosphatidyl-inositol 3'-kinase (PI3 kinase) signaling
pathway, can stimulate myogenesis and oppose the inhibitory action of FGF
(Kaliman et al., 1998;
Kaliman et al., 1996
;
Milasincic et al., 1996
;
Tamir and Bengal, 2000
;
Tureckova et al., 2001
). The
inhibition of sex muscle differentiation by EGL-15(neu*) provides
an in vivo system to test the involvement of the PI3 kinase signaling pathway
in muscle differentiation in C. elegans. PI3 kinase phosphorylates
PIP2 to generate PIP3, which in turn activates several downstream signaling
components (Cantrell, 2001
).
This activity is antagonized by the lipid-phosphatase PTEN, regulating the
amount of PIP3 generated (Leslie and
Downes, 2002
). The C. elegans PI3 kinase, AGE-1
(Morris et al., 1996
), also
functions within an insulin-signaling pathway and is opposed by the PTEN
ortholog, DAF-18 (Gil et al.,
1999
; Mihaylova et al.,
1999
; Ogg and Ruvkun,
1998
). Downstream components of this pathway include the
serine/threonine kinases PDK and AKT, encoded by the pdk-1, akt-1 and
akt-2 genes (Paradis et al.,
1999
; Paradis and Ruvkun,
1998
).
Based on the antagonistic effects of FGF and insulin on myogenesis in vertebrates, increases in the activity of the PI3 kinase signaling pathway would be predicted to suppress the effects of hyperactivated EGL-15 and restore muscle differentiation to soc-2; ayIs15 animals. Consistent with this hypothesis, loss-of-function mutations in daf-18 and gain-of-function mutations in pdk-1 and akt-1 all suppress the sex muscle differentiation defect of soc-2; ayIs15 animals (Fig. 7C-E; Table 2). Eliminating DAF-18/PTEN has the most dramatic suppression, restoring sex muscle differentiation in 100% of daf-18(nr2037) soc-2; ayIs15 animals. The gain-of-function mutations pdk-1(mg142gf) and akt-1(mg144gf) have weaker, but still dramatic suppressive effects (68% and 82.5%, respectively). Suppression of the differentiation defect in all of these strains is indicated by restoration of sex muscle morphology, muscle actin filament structure and the robust expression of Pmyo-3::GFP in the vulval muscles (Fig. 7).
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Discussion |
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The severe effect of hyperactivated EGL-15 on sex muscle development appears to be due primarily to the inhibition of muscle differentiation in this lineage. Although there are earlier defects within the M lineage, SMs are still generated in soc-2; egl-15(neu*) animals and undergo the normal number of rounds of proliferation to generate sex muscle precursors. SM descendants, however, fail to mature properly. Immunohistochemical analysis of wild-type EGL-15 expression revealed that EGL-15 is normally expressed in the undifferentiated cells of the SM lineage and becomes restricted to the four differentiated vm1s in the adult. In soc-2; egl-15(neu*) animals, however, many more cells express EGL-15 during adulthood. Furthermore, these cells remain morphologically immature, unlike differentiated muscle cells. These cells also fail to stain for F-actin and only weakly express Pmyo-3::GFP, both molecular markers of differentiated muscle cells. Taken together, these data indicate that hyperactivation of EGL-15 represses sex muscle differentiation.
EGL-15 hyperactivation also appears to be able to inhibit muscle
development more generally, as expression of EGL-15(neu*) in body
wall muscle precursor cells can also perturb the development of the body wall
musculature. Phlh-1::egl-15(neu*) transgenic
animals display a lumpy-dumpy phenotype that phenocopies hlh-1(0)
animals (Chen et al., 1994),
both of which display abnormally developed body wall muscles. The common lumpy
dumpy phenotype of these animals may reflect the negative regulation of HLH-1
by EGL-15 activation in body wall muscle precursor cells. Consistent with this
hypothesis, FGF signaling can negatively regulate MyoD function via inhibitory
phosphorylation by protein kinase C (Li et
al., 1992
). The restricted muscle defects observed in
Pegl-15::egl-15(neu*) transgenic animals is
probably due to high levels of egl-15 expression being confined to
muscle precursor cells within the M lineage. Although high levels of EGL-15
have not been observed in the body wall muscles, EGL-15 has been shown to play
a role in protein degradation in differentiated body wall muscles
(Szewczyk and Jacobson,
2003
).
Not only has the analysis of egl-15(neu*) phenotypes
provided insights into muscle development, it has also served to confirm and
extend our understanding of additional roles of EGL-15 in C. elegans.
FGF signaling through EGL-15 is thought to play an instructive role in guiding
the migrating SMs to their precise final positions
(Burdine et al., 1998;
DeVore et al., 1995
;
Goodman et al., 2003
). The
disruption of proper migration events by hyperactivation of the normal
signaling pathway has often been used as evidence for an instructive role for
the affected pathway (Duchek et al.,
2001
). The behavior of the SMs in egl-15(neu*)
animals is consistent with an instructive role of EGL-15 in SM migration. Our
results also fit the temporal series of events thought to lead to muscle
differentiation. Expression of egl-15(neu*) under the
control of the unc-54 promoter fails to confer the same lumpy-dumpy
phenotype in transgenic animals that is observed when the
hlh-1/CeMyoD promoter is used. Only cells well along their commitment
to a muscle fate begin to express the UNC-54/MYO-4 myosin heavy chain
(Ardizzi and Epstein, 1987
;
Miller et al., 1983
). Unlike
uncommitted muscle precursor cells, these cells have apparently progressed
sufficiently along the myogenic pathway so as to be insensitive to the
inhibitory signal resulting from hyperactive EGL-15 signaling. Finally,
analysis of egl-15(neu*) animals has revealed a potential
new role for EGL-15 signaling in influencing cell division polarity in the
early M lineage. In the wild type, the division planes for cells in the M
lineage are spatially determined (Sulston
and Horvitz, 1977
). Hyperactivation of EGL-15 disrupts the
polarity of these divisions, and the cells divide along improper division
planes. It will be interesting to use egl-15(neu*) to
probe the mechanisms that determine the polarities of these divisions.
A model for studying myogenesis
The phenotypes of egl-15(neu*) are due to dramatic
constitutive activation of EGL-15, and, therefore, do not necessarily reflect
the normal roles of EGL-15. Nonetheless, the effects of
egl-15(neu*) on sex muscle differentiation are an accurate
reflection of a normal role of FGF signaling during vertebrate myogenesis. In
cell culture, activation of the FGF receptor inhibits myoblast differentiation
(Clegg et al., 1987;
Spizz et al., 1986
;
Templeton and Hauschka, 1992
;
Vaidya et al., 1989
). This
effect of FGF signaling is antagonized by the PI3 kinase signaling pathway,
which functions to promote myogenesis. Overexpression and activation of the
PI3 kinase signaling components PDK1 and AKT1 can result in myoblast
differentiation even in the presence of an FGF signal that normally blocks
muscle differentiation (Kaliman et al.,
1996
; Milasincic et al.,
1996
; Tamir and Bengal,
2000
; Tureckova et al.,
2001
). In C. elegans, signaling through the PI3 kinase
pathway can be increased either by loss of DAF-18/CePTEN or via
gain-of-function alleles of pdk-1 or akt-1
(Gil et al., 1999
;
Mihaylova et al., 1999
;
Ogg and Ruvkun, 1998
;
Paradis et al., 1999
;
Paradis and Ruvkun, 1998
).
Similar to the antagonism between FGF and PI3 kinase signaling observed in
cultured myoblasts, all of these mutations can suppress the sex muscle
differentiation defect in egl-15(neu*) animals. Thus, the
effects of EGL-15(neu*) on sex muscle differentiation parallel the
effects of mammalian FGF receptor activation in cultured myoblasts and can
serve as a good model for understanding the complex interplay of signaling
pathways in myogenic processes.
The myogenic function of the PI3 kinase pathway in C. elegans
appears to be mediated by an effector other than the DAF-16 transcription
factor normally associated with PI3 kinase signaling in worms. Activation of
PI3 kinase signaling using a null allele of daf-18/CePTEN can cause
dramatic suppression of the inhibition of sex muscle differentiation because
of egl-15(neu*). PI3 kinase signaling normally inhibits
the function of HNF-Forkhead transcription factors that transduce the effects
of the upstream signaling pathway (Lin et
al., 2001; Ogg et al.,
1997
; Tang et al.,
1999
). Thus, loss of these factors often mimics activation of the
signaling pathway. The suppression of the muscle differentiation defect by
daf-18(nr2037) is not mimicked by eliminating DAF-16, the forkhead
transcription factor that functions as the target of PI3 kinase signaling in
other known events in C. elegans
(Lin et al., 2001
;
Ogg et al., 1997
). Thus, our
data suggest that other targets of the PI3 kinase pathway function in the
process of sex muscle differentiation, and that signaling specificity in this
pathway is likely to be derived from tissue specific expression of
AKT-regulated factors. Likely candidates include the Foxo transcription
factors (Arden and Biggs, 2002
;
Hope et al., 2003
) and
TOR/p70S6 kinase signaling pathways, which have been shown to be regulated by
AKT and to be important for myoblast differentiation in vivo
(Conejo et al., 2002
;
Hribal et al., 2003
).
Separation of EGL-15 function
FGFs can stimulate many distinct biological processes, and multiple
mechanisms are used to specify the consequence of their activation
(Szebenyi and Fallon, 1999).
FGF signaling in C. elegans regulates a number of processes
(Burdine et al., 1998
;
DeVore et al., 1995
;
Szewczyk and Jacobson, 2003
),
and the mechanisms that determine which process is triggered are beginning to
be revealed.
One major determinant of signaling specificity is tissue-specific
expression. EGL-15 expression in the hypodermis regulates fluid balance
(Huang and Stern, 2004),
whereas expression in the M lineage controls a number of aspects of sex muscle
development (Burdine et al.,
1998
). The restricted normal expression of egl-15 to the
M lineage apparently confines the dramatic effects of
egl-15(neu*) to the sex muscles while leaving body wall
muscle development intact. Even the timing of expression within a cell lineage
is crucial for specifying potential outcomes: early expression of
EGL-15(neu*) in muscle precursor cells has dramatic effects on
myogenesis, while its expression in committed muscle cells does not.
Alternative splicing is a second important mechanism used to increase
receptor diversity and potential for signaling specificity
(Green et al., 1996). In
C. elegans, egl-15 is alternatively spliced to yield two isoforms,
EGL-15(5A) and EGL-15(5B). These isoforms can be assigned to different
physiological functions of EGL-15. The 5B isoform is required to maintain
fluid homeostasis, while the 5A isoform is essential for chemotaxis of the
M-derived sex myoblasts (Goodman et al.,
2003
). The effects of hyperactivated EGL-15 are also specified in
large part by this alternative splicing event; hyperactivation of 5B results
in the dramatic Clear phenotype, while hyperactivation of 5A has effects that
are predominantly confined to the M lineage.
Differences in signaling pathway components or their activity thresholds
(Tan and Kim, 1999) also can
play a crucial role in the specification of signaling outcomes
(Boilly et al., 2000
). The
effects of a soc-2 mutation on the processes affected by EGL-15
hyperactivation is a potent illustration of the importance of this mechanism.
The phenotypic specificity of a hypomorphic soc-2 mutation revealed
the effects of hyperactive EGL-15 signaling on sex muscle development.
Delineating the EGL-15 pathway used in myogenesis will be an important step
towards a better understanding of EGL-15 signaling specificity mechanisms.
Moreover, the amenability of the sex muscle phenotype to systematic genetic
analysis could also provide a broader understanding of how the FGF pathway
interacts with the other signaling pathways required for functional muscle
formation.
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ACKNOWLEDGMENTS |
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