1 Developmental Biology Program, Sloan-Kettering Institute, Memorial
Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
2 Departments of Developmental Biology and Genetics, Howard Hughes Medical
Institute, Beckman Center, B300, 279 Campus Drive, Stanford University School
of Medicine, Stanford, California 94305-5329, USA
Author for correspondence (e-mail:
m-baylies{at}ski.mskcc.org)
Accepted 21 August 2003
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SUMMARY |
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Key words: Myogenesis, Founder cells, Myoblast fusion, phyllopod, Toll, Microarrays
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Introduction |
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Diversity in muscle identity begins in Drosophila with the
specification of two types of myoblasts: founder cells (FCs), which contain
information required for morphogenesis of a given muscle; and fusion-competent
myoblasts (FCMs), which fuse to a FC and become entrained to a particular
muscle program (Bate, 1990;
Dohrmann et al., 1990
).
Production of the two myoblast types requires a combination of signals from
the ectoderm and mesoderm (reviewed by
Baylies and Michelson, 2001
;
Frasch, 1999
). In the dorsal
mesoderm, two secreted signals, Wingless (Wg) and Decapentaplegic (Dpp),
converge to define a region competent to respond to inductive signals mediated
by Ras signaling. Localized Ras activation in dorsal cells instructs clusters
of myoblasts to adopt the FC fate (Fig.
2A) (Buff et al.,
1998
; Carmena et al.,
1998a
). The FC fate is restricted to one cell within each cluster,
the muscle progenitor, by a process of lateral inhibition mediated by Notch
/Delta signaling (Bate et al.,
1993
; Carmena et al.,
1995
; Corbin et al.,
1991
; Giebel,
1999
) and the activity of Argos, a diffusible inhibitor of the
Drosophila Epidermal Growth Factor Receptor (Egfr, previously known
as DER) (Carmena et al.,
2002
). Cells receiving the Delta signal from a neighboring muscle
progenitor are determined to become FCMs. Thus, during specification of muscle
progenitors from clusters of equivalent myoblasts, the Ras pathway induces the
muscle progenitor fate, whereas the Notch pathway promotes the FCM fate.
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After FCs and FCMs are specified, fusion ensues under the control of
cell-cell interaction regulators. Cell surface recognition proteins are
produced specifically in the founder myoblast, which mediates fusion to FCMs.
The recognition proteins include Kin of Irre/Dumbfounded (Kirre/Duf)
(Ruiz-Gómez et al.,
2000), Roughest/Irregular Chiasm (Rst/IrreC)
(Strunkelnberg et al., 2001
)
and the intracellular adapter protein Rolling pebbles/Antisocial (Rols/Ants)
(Chen and Olson, 2001
;
Menon and Chia, 2001
;
Rau et al., 2001
). FCMs, which
are produced in response to the activation of Notch, appear to have their own
distinct differentiation program. FCMs produce specific proteins required for
fusion to FCs, such as Sticks and Stones (Sns)
(Bour et al., 2000
), Hibris
(Hbs) (Artero et al., 2001
;
Dworak et al., 2001
) and the
transcriptional regulator Lame Duck (Lmd)
(Duan et al., 2001
;
Furlong et al., 2001
;
Ruiz-Gómez et al.,
2002
). Although all these proteins have specific functions within
either FCs or FCMs, a comprehensive understanding of the fusion process is
clearly lacking.
The specific combination of inputs that a given FC receives results in the
production of the unique set of molecular determinants that gives each muscle
fiber its shape, size and connection pattern. Transcription factors, such as
Krüppel (Kr) or Even-skipped (Eve), are produced in specific FCs in
response to the signals. These transcription factors, through as yet unknown
mechanisms, regulate the attributes of each muscle
(Carmena et al., 1998a;
Halfon et al., 2000
;
Ruiz-Gómez et al.,
1997
). The number of known molecular determinants is substantially
less than the number of muscle fiber variants, which suggests that more
molecular determinants remain to be discovered.
Here, we report the identification of additional genes that regulate the
properties and functions of FCs and FCMs, using a genetic strategy coupled to
a cDNA microarray approach. We exploited the response of the somatic mesoderm
to Ras and Notch signaling to specifically enrich embryos in FCs, which are
Ras-dependent, or in FCMs, which are Notch-dependent. FCs and FCMs are
low-abundance cell types, so the overexpression experiments were carried out
in Toll10b mutant embryos. Cells of
Toll10b embryos differentiate primarily as somatic
mesoderm, and the embryos are relatively enriched for FCs and FCMs
(Casal and Leptin, 1996;
Leptin et al., 1992
;
Ray et al., 1991
) (this work).
Newly identified genes predicted to be enriched in FCs or FCMs were confirmed
using northern blot analysis and in situ hybridization, providing validation
for our approach. We investigated the phenotype of a selected set of newly
identified genes and found that although some (e.g. phyllopod,
asteroid) are crucial for the specification of particular muscles, others
(e.g. tartan) are involved in the elaboration of specific muscle
morphologies.
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Materials and methods |
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Fly stocks for newly identified genes
A P element insertion, EfTuML4569, in the mitochondrial
Elongation factor Tu gene (CG6050) was used. Embryos homozygous for
EfTuML4569 are lethal, whereas embryos that are
transheterozygous for the P insertion and a deficiency from the same region
[Df(2R)CX1] reveal a partial fusion block and a gut phenotype.
Gs11 is a lethal EMS-induced mutation in glutamine
synthetase 1 (CG2718), which, in trans with a deficiency from the region
(Df(2L)PM1), reveals several subtle morphological defects. For example, the
dorsal muscles were sometimes split in two, the DO2 muscle was thinner than
usual and the dorsal muscles show aberrant attachment to the apodemes. Because
of our characterization of the nearby gene hibris, we initially had
available deficiency combinations that removed parcas and two
additional predicted genes that are not expressed in the embryo. In these
mutant embryos, we detected a muscle phenotype, including muscle losses and
attachment defects. A mutation in parcas
(Sinka, et al., 2002), in
trans with our deficiencies, has a similar muscle phenotype. The complete
analysis of parcas function during muscle development will be
published elsewhere. In the case of CG7212, the gene trap insertion
BG02608 is a P insertion 27 bp downstream of the start site and is
predicted to be a null allele. Although the initial specification of the
muscles appeared normal by FC marker analysis, the LT1-4 and DT1 muscles, for
example, were frequently shorter (data not shown). Muscles in CG7212
mutants also had defects in their growth toward the epidermal attachment sites
and displayed abnormal shapes (e.g. LT4 muscles). We propose the name
cadmus (cdm; after the mythological figure that changed his
shape into a snake) for the predicted gene CG7212. The muscle
phenotype detected in BG02608 homozygous mutant embryos was the same
as this allele over a deficiency from the relevant region. The percentage of
mutant hemisegments was calculated by counting between 65 (ast) to
120 (trn, phyl) myosin-stained hemisegments.
Molecular biology
RNA was extracted from appropriate samples using Tri-Reagent (Sigma), and
polyA+ RNA was purified with Oligotex mRNA spincolumns (Qiagen).
Northern blots were processed following manufacturer recommendations for
digoxigenin-labeled probes (Roche Molecular Biochemicals). Probes employed in
the northern blots with the corresponding genes in parenthesis were: GH09755
(parcas), SD10254 (CG4136), GH20973 (gol), CK00397
(dei), CK00321 (CG8503), GH20549 (CG6024), pPhyl/BS
(phyl) (Chang et al.,
1995; Dickson et al.,
1995
), LD36757 (CG17492), GH10871 (trn) and
CK00552 (nidogen). DNA was sequenced at the Cornell University DNA
sequencing facility (Ithaca, NY).
Histology techniques
Immunocytochemistry in embryos was performed as described by Rushton et al.
(Rushton et al., 1995), with
the following modifications. Antibodies were pre-absorbed against fixed
wild-type embryos, and in combination with the TSA system (PerkinElmer Life
Sciences) or a hot fixation protocol
(Rothwell and Sullivan, 2000
),
as indicated by the acronym TSA or HF, respectively. Antibody dilutions were:
anti-Mhc (1:1000) (Kiehart and Feghali,
1986
), anti-ß-Gal (1:2000; Promega), anti-Slouch (1:200),
anti-Vg (1:100), anti-Sns (1:1000; HF; TSA)
(Bour et al., 2000
),
anti-FasIII (1:100) (Patel et al.,
1987
), anti-Twist (1:5000; provided by S. Roth), anti-Tinman
(1:2500; TSA), anti-Crumbs (1:50), anti-Single-minded (1:1000; HF; TSA;
provided by S. Crews), anti-22C10 (1:20), anti-Trachealess (1:2000; HF; TSA),
anti-Wg (1:200) and anti-Htl (1:2000; provided by A. Michelson). Antibodies
against FasIII, 22C10, Trachealess and Wg were obtained from the Developmental
Studies Hybridoma Bank, University of Iowa. Fluorescent immunohistochemistry
with anti-Ubx (1:300; TSA) and anti-Trn (pre-absorbed 1:900; TSA)
(Chang et al., 1993
) was
achieved using streptavidin FITC. Anti-Kr (1:500; provided by J. Reinitz) was
detected with the TSA Fluorescence System (PerkinElmer Life Sciences).
Anti-Ase was used pre-absorbed at a 1:2500 dilution and detected with
Cy3-conjugated streptavidin. Biotinylated secondary antibodies were used in
combination with Vector Elite ABC kit (Vector Laboratories, CA). Specimens
were embedded in Araldite and images captured using an Axiocam camera (Zeiss)
using Adobe Photoshop software. RNA localization was detected using
digoxigenin-labeled RNA probes as described
(O'Neil and Bier, 1994
).
Fluorescent in situ hybridizations to detect dei, gol, nidogen and
phyl transcripts were performed using digoxigenin-labeled RNA probes
under standard conditions (50% formamide at 65°C overnight). Following
repeated washing at 65°C, the embryos were rehydrated in step wise fashion
and incubated in blocking solution [0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl, 0.5%
blocking reagent; NEN) for 1 hr at 22°C. The embryos were then incubated
at 4°C overnight in 250 µl blocking solution with a 1:2000 final
concentration of anti-digoxigenin-POD (Roche), pre-adsorbed against fixed
embryos. Following 12x30 minute washes, the embryos were developed
with the TSA-Plus Fluorescence Palette system (NEN), using a 3 minute
incubation with a 1:50 dilution of fluorescein amplification reagent. After
4x15 minute washes, the embryos were incubated with a rabbit
anti-ß-Gal antibody at 4°C overnight to detect the rp298
driven lacZ expression. This reaction was visualized using a
biotinylated anti-rabbit secondary antibody and streptavidinrhodamine (1:250;
Jackson Laboratories).
Microarray methods
The microarrays used for the analysis contained over 8500 ESTs
corresponding to 4949 unique genes plus a variety of controls. To generate
microarray probes, 2.5 µg polyA+ RNA was incubated with 4 µg
of an equal ratio mixture of random hexamers and oligo dT, in a final volume
of 15 µl at 70°C for 10 minutes. The reaction was then placed on ice
and 6 µl 5x first strand buffer (Gibco BRL), 3 µl 0.1 M DTT, 3
µl Cy3 or Cy5-dUTP dye, 0.6 µl dNTPs [25 mM dATP, 25 mM dCTP, 25 mM dGTP
and 10 mM dTTP] and 2 µl SuperScript II reverse transcriptase (Gibco BRL)
was added. The reaction was placed at 42°C for 2 hours. RNA base
hydrolysis was used to stop the reaction by adding 1.5 µl 1 M NaOH, 20 mM
EDTA and placing at 65°C for 10 minutes. The pairs of Cy3 and Cy5
reactions were pooled together with 60 µl water and 15 µl NaAc (pH 5.2).
Unincorporated dyes were removed by adding 500 µl Qiagen PB buffer and
purifying using the Qiaquick purification system according to manufacturer's
instructions. The reactions were eluted off the Qiaquick mini columns using
two 30 µl EB buffer washes. The samples were concentrated using a
Microcon-30 spin column. For array hybridization, the sample volume was
adjusted to 10.4 µl and 3 µl 20xSSC, 1.2 µl 10 mg/ml
polyA+ RNA and 0.4 µl 10% SDS were added. The sample was heated
to 100°C for 2 minutes, briefly spun and immediately added to the array.
The arrays were hybridized in a chamber with 3xSSC at 65°C for 12-14
hours. Following hybridization, the array was successively washed in three
solutions: (1) 1xSSC, 0.03% SDS; (2) 0.2xSSC; and (3)
0.05xSSC. Following drying by centrifugation at 129 g
for 5 minutes, the slides were immediately scanned using an Axon Scanner. For
each of the two experimental conditions, three independent embryo collections
(aged 5-9 hours AEL) and hybridizations were used.
Statistical analysis was performed as described
(Tusher et al., 2001). The
fold difference was defined as the mean of ratios of activated Ras/activated
Notch conditions from three independent hybridization experiments. Genes were
considered to be differentially expressed whenever one of the following
conditions were met: (1) a fold difference of 2 or above (Ras conditions) or
0.5 and below (Notch conditions) in three independent hybridization
experiments; or (2) significance by SAM statistical analysis
(Tusher et al., 2001
) with a
fold difference of 1.8 or above (Ras conditions) and 0.6 and below (Notch
conditions), when data from at least two independent hybridization experiments
were available. Curated information from Drosophila genes was
obtained from
http://www.flybase.org/
and expression patterns from the BDGP expression pattern project at
http://www.fruitfly.org/cgibin/ex/insitu.pl/.
Published array databases for mesoderm
(Furlong et al., 2001
) or
developmental stages (Arbeitman et al.,
2002
)
(http://www.fruitfly.org/cgi-bin/ex/basic.pl)
were also analyzed to confirm mesodermal and/or embryonic expression.
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Results |
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Signaling pathways required for mesoderm differentiation include Wg,
Hedgehog (Hh), Dpp, Notch and Ras, all of which we evaluated in
Toll10b mutant embryos. dpp and hh
transcripts were absent from Toll10b embryos (not shown),
with the exception of transient, early dpp expression at the anterior
and posterior ends of syncytial blastoderm embryos
(Ray et al., 1991) (data not
shown). Toll10b mutant embryos stained with an anti-Htl
antibody had clusters of Htl-positive cells in the mesoderm, reminiscent of
wild-type controls (Fig. 1Y-Z)
(Michelson et al., 1998a
).
Anti-Wg staining revealed stripes of expression
(Fig. 1AA-BB), in contrast to
the wild-type situation in which Wg is normally undetectable in the mesoderm
at this time (Baylies et al.,
1995
). Successful Ras and Notch signaling was inferred from
biological responses in Toll10b mutant embryos expressing
activated Ras or Notch (Fig. 2;
see below). Hence, Wg, Ras and Notch signaling pathways that are crucial to FC
specification are active in Toll10b mutant embryos.
Toll10b mutant myoblasts respond to Ras and Notch
signaling like their wild-type counterparts
For Toll10b embryos to be useful for our screen, FCs
and FCMs must be specified and responsive to Ras and Notch signaling despite
the altered tissue composition. Two genes expressed in FCs, slouch
(Dohrmann et al., 1990) and
vestigial (vg) (Bate et
al., 1993
), marked groups of myoblasts in
Toll10b mutant embryos, indicating that FC specification
occurs (Fig. 2B,E). FC genes
that are normally expressed in dorsal cells (e.g. eve or
runt) were not detected, presumably because of the absence of
ectoderm-derived dpp (Halfon et
al., 2000
) (data not shown). Large numbers of FCMs developed in
Toll10b mutants, as shown by the presence of the
FCM-specific protein Sns (Fig.
2H).
When activated forms of Notch or Ras were expressed using the twist-Gal4 driver in a Toll10b mutant, the number of FCs decreased (Fig. 2C,F) or increased (Fig. 2D,G), respectively, compared with Toll10b embryos. Moreover, the FCM marker sns showed increased expression when FC markers decreased: activated Notch increased sns expression and Ras activation reduced sns expression (Fig. 2I,J). The dramatic changes in FC and FCM numbers when Ras or Notch were activated in mesoderm enriched embryos made it possible to analyze changes in transcript levels in normally scarce muscle progenitor cells.
Gene expression profile of Toll10b embryos with Ras or
Notch activated in the somatic mesoderm
RNA from 5- to 9-hour Toll10b embryos expressing
activated Ras (FC-enriched) or activated Notch (FCM-enriched) were collected
and hybridized to cDNA arrays containing 4988 Drosophila genes [for details,
see Materials and methods, and Furlong et al.
(Furlong et al., 2001)]. The
differences between expression levels for each gene under the two conditions
were measured as the value of activated Ras divided by the value for activated
Notch. We defined the Fold difference (F;
Table 1) as the mean of the
ratios from three independent hybridization experiments. Thus a fold
difference of 1 indicates that the gene in question did not appear to be
regulated differently by either Ras or Notch (hence was 'equally expressed' in
both FCs and FCMs). Values greater than 1 result from genes with transcripts
'enriched in FCs', whereas values less than 1 indicate genes encoding
transcripts 'enriched in FCMs'.
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The second control group contains muscle identity and fusion genes already
known to be expressed only in one type of myoblast. Three genes previously
shown to be necessary for FC specification, stumps, htl and big
brain, were identified (Table
1) (Corbin et al.,
1991; Imam et al.,
1999
; Michelson et al.,
1998b
; Vincent et al.,
1998
). Both htl and stumps are specifically
expressed in FCs, validating our approach. Genes required for myoblast fusion,
including hbs and sallimus/DTitin (sal),
had increased transcript abundance under activated Notch conditions.
hbs is expressed in FCMs (Artero
et al., 2001
; Dworak et al.,
2001
). Expression of sal occurs in both types of
myoblasts, yet transcripts are more abundant in FCMs than in FCs
(Menon and Chia, 2001
).
Several known genes that were expected to be differentially expressed, such as
kirre/duf, rst/irreC, slouch, Kr, vg or
apterous, were not present on the array whereas others, such as
sns, rols/ants and connectin, showed altered
expression as expected but did not meet our stringent statistical requirements
(see Table S1 at
http://dev.biologists.org/supplemental/).
Other known genes, such as eve, were not expected to be
differentially expressed because their expression requires Dpp signaling,
which is absent from Toll10b embryos.
Taken together, the controls indicated that the screen was able to identify genes differentially expressed between FCs and FCMs. Below we highlight several classes of genes involved in different facets of muscle morphogenesis. In addition to our stringent statistical requirements, the genes discussed have the expected biological properties, such as expression in the proper cells and/or relevant mutant phenotypes (Table 1; Figs 3, 4, 5, 6).
Transcription factors expressed specifically in FCs or FCMs
The discovery of transcription factors expressed specifically in subsets of
FCs (Dohrmann et al., 1990)
and in FCMs (Duan et al.,
2001
; Furlong et al.,
2001
; Ruiz-Gómez et
al., 2002
) suggests that individual FCs and FCMs have different
potentials. We have identified additional transcription factors that are
enriched in FCs and FCMs. delilah (dei), which encodes a
basic helix-loop-helix (bHLH)-containing protein
(Armand et al., 1994
), is
expressed within FCMs. The differential transcription of dei was
confirmed by using northern blot analysis
(Fig. 3) and in situ
hybridization (Bouchard and Cote,
1993
) (Fig. 4A-C).
dei transcript levels decline in the somatic mesoderm as fusion
proceeds. dei expression did not recur in developing muscles,
suggesting that it is required to control some aspect of FCM identity before
fusion. Two other FCM-enriched transcription factors were identified in the
screen, the bHLH containing protein mß of the E(spl) complex and
the homeobox-containing gene CG4136 (see blot in
Fig. 3). The involvement of the
E(spl) complex in muscle development has been reported previously
(Bate et al., 1993
;
Corbin et al., 1991
), but the
particular member of the complex that is involved remained unknown. Our data
suggest that the mß transcript is one of the members of the
E(spl) complex required for muscle development.
Two transcription regulators, asense (ase) and
Ultrabithorax (Ubx), were induced under activated Ras
conditions and predicted to be active in FCs. Ase, an achaetescute
(ac-sc) complex member, is a bHLH transcription factor that functions as
a proneural gene in bristle development. During muscle development in
mid-stage 12 embryos, Ase was transiently expressed in Kr-positive FCs
(Fig. 4G-I). The homeotic gene
Ubx was predicted to be expressed in FCs based on previous reports
that showed that Ubx is involved in muscle pattern diversification
(Michelson, 1994). Confocal
analysis confirmed that Ubx was expressed in FCs
(Fig. 4J-L).
Signaling-related molecules enriched in FCMs or FCs
Seven genes that encode proteins predicted to be involved in cell signaling
were enriched under activated Notch conditions and therefore predicted to be
active in FCMs (Table 1). Three
of these, parcas, asteroid (ast) and goliath (gol),
encode novel molecules. parcas is the Drosophila homolog of
the human SAB gene. SAB protein inhibits the auto- and
transphosphorylation of BTK, a tyrosine kinase crucial for B cell development
(Yamadori et al., 1999).
Regulation of parcas under our experimental conditions was confirmed
by northern blot (Fig. 3).
parcas is transcribed in embryos during somatic muscle development.
Embryos in which parcas function has been removed have muscle
phenotypes (Beckett et al.,
2002
) (see Materials and methods). Ast is a novel 815 amino acid
protein. Expression and phenotypic analyses of ast are presented in
detail below. A third FCM signaling gene, gol, encodes a RING-finger
protein. Its differential expression was confirmed by northern analysis
(Fig. 3) and its FCM expression
was confirmed by in situ hybridization
(Fig. 4D-F). The
Xenopus Gol homolog GREULI functions as an E3 ubiquitin ligase. It
has been proposed that GREULI modulates signal transduction pathways required
for neuralization of ectoderm (Borchers et
al., 2002
).
Three putative signaling proteins that were previously characterized in
other tissues had transcripts enriched in FCs, suggesting roles in muscle
development: RhoGEF3, polychaetoid and phyllopod (phyl).
RhoGEF3 encodes the Drosophila ortholog of the human protein
PEM2, a GEF regulator of Rho activity that is transcribed in muscle cells
during morphogenesis (Hicks et al.,
2001). Polychaetoid (Pyd) is a cytoplasmic PDZ and SH3
domain-containing protein required for dorsal closure
(Takahashi et al., 1998
), and
for proper segregation of sensory organ precursors from proneural clusters
(Chen et al., 1996
). Phyllopod
(Phyl) is discussed in more detail below. These findings highlight possible
mechanisms through which signals are conveyed to the cytoskeleton to elaborate
muscle morphology.
Myoblast fusion and cell adhesion
Myoblast fusion between FCs and FCMs occurs through the interaction of
proteins that are produced in one or the other muscle cell type, for example
Kirre/Duf in FCs and Sns in FCMs (Bour et
al., 2000; Ruiz-Gómez
et al., 2000
). Our data identified three genes known to be
required for myoblast fusion that are enriched in FCMs: hibris, blown
fuse and sallimus/DTitin. A novel gene induced by Ras signaling,
CG17492 (Table 1),
encodes a protein predicted to contain domains involved in protein-protein
interactions also found in the adaptor protein Ants/Rols. Our in situ
hybridization data indicate that it is strongly expressed in the somatic
mesoderm (data not shown).
Work by Chen and Olson (Chen and Olson,
2001) has shown that two forms of Kirre/Duf, a cleaved and an
uncleaved version, can be detected when Kirre/Duf is expressed in S2 cells.
They hypothesize that the cleaved form functions as the attractive signal,
which emanates from FCs. Because of its redundancy in structure and function,
Rst/IrreC has been proposed to behave similarly
(Dworak and Sink, 2002
).
Although the enzyme that cleaves Kirre/Duf is unknown, we have found a
serine-type endopeptidase and two serine protease inhibitors that were induced
under activated Notch conditions and are therefore predicted to be expressed
in FCMs. FCs may have their own endopeptidase system: tequila, a gene
encoding a serine-type endopeptidase, was found to be enriched under Ras
conditions and is therefore predicted to be in FCs
(Table 1). It is tempting to
suggest that these protease activities and inhibitors are involved in
regulating production of the signaling-competent forms of Kirre/Duf and
Rst/IrreC, and/or its diffusion among FCMs.
Two genes putatively involved in cell adhesion, tartan
(trn) and the predicted gene CG10275, were enriched in
activated Ras conditions. Trn, a Leucine-Rich Repeat (LRR)-containing
transmembrane protein, contributes to the formation of the dorsoventral
boundary in the developing wing (Gabay et
al., 1996; Milan et al.,
2001
). The expression of tartan in FCs and the muscle
phenotype of tartan mutants are described below.
In parallel with cell adhesion, substrate adhesion may also guide muscle
morphogenesis. We found that transcripts of Drosophila nidogen, which
encode an extracellular matrix protein that co-localizes with laminin in the
basement membranes of muscles (Kumagai et
al., 1999), are induced in FC-enriched embryos. Northern blot
analysis confirmed that nidogen was expressed at higher levels under
activated Ras conditions (Fig.
3), and fluorescent in situ hybridization showed that the
transcript is specifically localized in a subset of FCs
(Fig. 4P-R).
Genes with uncertain roles during muscle development
Although all 83 genes that met our stringent statistical criteria are shown
in Table 1, some of the genes
are not yet clearly linked to a role in wild-type muscle development by
expression or phenotype. We therefore listed these genes as having 'specific
role in muscle development uncertain'
(Table 1). However, we
emphasize that these genes may have been correctly identified as responsive to
the genetic conditions used in our screen, which may influence genes in
addition to muscle genes.
During the screen validation process we have further classified the genes
with no clear mesoderm function into subgroups. For example, wingless,
CG6024 and CG4136 form a group because they are not normally
expressed in wild-type somatic mesoderm but their differential expression has
been confirmed by northern blot (Fig.
3). This group of genes may normally be repressed in the mesoderm
by a regulator that is absent in the Toll10b background. A
second group consists of genes that are normally expressed in tissues other
than somatic mesoderm that are still present in Toll10b
embryos. For example, bangles and beads, CG1124 and CG14989
are expressed in the mesectoderm during the 5-9 hour developmental window used
in our experiments (BDGP expression data, and data not shown). Similarly,
other genes expressed in the endoderm, blood progenitors or other tissues
present in Toll10b embryos, may be subject to Notch and/or
Ras regulation. A third group is comprised of two repetitive retrotransposons:
gypsy and 412. Retrotransposable elements are flanked by
Long Terminal Repeats with promoter activity
(Levin, 2002). For some copies
of the transposons, these promoters might act as reporters for nearby
differentially expressed genes, and thus appear specifically enriched in one
of the conditions. The analyses to date, therefore, lead us to expect that the
true false-positives emerging from the screen will be a small minority of the
total.
Phenotypes associated with new FC/FCM regulatory genes
The differential expression of a number of genes identified in the screen
were confirmed by northern blot and/or immunostaining/fluorescent in situ
hybridizaton. We assayed a subset of genes from the screen to determine
whether these genes have an essential role in normal muscle development. We
obtained available loss-of-function mutants in ast (a FCM-enriched
signaling-related protein), trn (a FC-enriched cell adhesion
molecule) and phyl (a FC-enriched signaling-related molecule) (see
Materials and methods). In each case, mutant embryos showed defects in the
somatic musculature, ranging from aberrant muscle morphologies and partial
fusion blocks (trn), to missing/duplicated muscles (phyl,
trn and ast). We discuss the phenotypes of these three genes in
more detail below.
Asteroid, a FCM signaling-related protein
Mutations in ast dominantly enhance Star mutations, and
also enhance the Ellipse mutation EgfrE1 in the
Drosophila eye. ast transcripts have been detected in the
somatic mesoderm up to stage 12 (Kotarski
et al., 1998). Although we have been unable to detect ast
expression in embryos by in situ hybridization, embryos carrying mutations in
ast have defects in the development of a subset of the somatic
muscles, particularly LL1 and DO4 (Fig.
5B). DO4 is affected in approximately 88% of the hemisegments,
whereas LL1 is affected in 10% of the hemisegments. Founder cell
specification, as judged by the FC marker Kr, which is expressed in the
founder cell for LL1, appears normal in ast mutant embryos (data not
shown).
Tartan, a FC-enriched cell adhesion protein
Trn is an Egf signaling transcriptional target that organizes the specific
affinities of cells in the dorsal compartment of the wing disc
(Gabay et al., 1996;
Milan et al., 2001
). We have
confirmed that, in the embryonic mesoderm, Trn is expressed only in FCs
(Fig. 4M-O). Homozygous
trn mutant embryos had abnormal somatic muscle morphology, with 60%
of the hemisegments showing some obvious muscle defects. Muscles such as LT1-4
and DT1 had aberrant shapes and attachments
(Chang et al., 1993
)
(Fig. 5C). Although anomalous
in shape, these muscles contained several nuclei suggesting that the fusion
process was not impaired in trn mutant embryos. Muscle losses and
gains were also detected for LT1-4, despite wildtype expression of the FC
marker Kr (data not shown). Although we show a lateral view of a stage 16
embryo in Fig. 5, all three
muscle groups - dorsal, lateral and ventral - are affected in trn
mutant embryos.
Phyllopod, a FC-enriched signaling related protein
phyl transcripts were enriched under activated Ras conditions,
predicting FC-specific expression for phyl. Expression of
phyl was confirmed in a subset of FCs in the somatic mesoderm
(Fig. 6A-C), and in the
visceral mesoderm (Fig. 6D-F).
Phyl, a novel adaptor protein, was already known to be required for
determination of photoreceptor and external sensory cell fates
(Li et al., 1997;
Pi et al., 2001
). Together
with Seven-in-absentia (Sina), Phyl promotes photoreceptor differentiation by
targeting the transcriptional repressor protein Tramtrack for degradation
(Dickson, 1998
;
Tang et al., 1997
), by acting
as part of an E3 ubiquitin protein ligase complex
(Li et al., 2002
).
We found that phyl is a crucial regulator of somatic muscle differentiation. Loss of phyl caused reproducible loss of a subset of the somatic muscles, including muscles LL1 and DO4 (Fig. 6I). LL1 was lost in approximately 52% of the hemisegments analyzed. To address whether phyl is required for specification of FCs, we examined Kr expression in phyl null embryos. Kr expression was often missing or at lower levels in approximately 26% of the LL1 FC, whereas expression of Kr and other FC markers in other FCs was normal (Fig. 6J). These data suggest that although Phyl is required for the specification of a subset of FCs, perhaps through the maintenance of FC determinants such as Kr, other FC identity genes, which work in combination with Kr, may also be targets.
Ectopic expression of phyl with the GAL4/UAS system resulted in alterations in muscle identities. For example, LL1 was occasionally affected by phyl overexpression, and DT1 and LT4 consistently had altered morphology. DT1 failed to attach normally, resulting in a 'ball of muscle', like that seen in myospheroid mutant embryos. LT4 failed to grow dorsally, which resulted in a smaller muscle (Fig. 6K). The specification of the Kr-positive LT4 FC appeared normal in these embryos (Fig. 6L), in keeping with detection of LT4, albeit in abnormal form. In conclusion, these data indicate a previously unknown role for Phyl during muscle specification and morphogenesis.
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Discussion |
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Various tests were applied to ascertain the validity of our results. Available databases (see Materials and methods) were analyzed to find evidence that the known and predicted genes in Table 1 were expressed at the correct time and place (Table 1, Biology). In addition, northern analysis with eleven genes tested the reliability of our microarray detection and selection criteria (Fig. 3); the results from all genes tested agreed with the array data.
A Toll10b sample on the northern blots allowed us to
ascertain why a gene is enriched in a particular condition. For example, in
the case of FC enriched genes, the signal in the Ras and
Notch lanes can be compared with Toll10b alone to
determine whether the Ras/Notch ratio for a gene is due to activation by Ras
or repression by Notch. Those genes in
Table 1 that are 'enriched
under Notch conditions', for example, could reflect a variety of transcription
mechanisms that would result in a ratio of less than 0.6. By northern
analysis, we find many of the 'Notch-regulated' genes, and hence the predicted
FCM genes, are repressed by Ras signaling and slightly activated by Notch. As
a case in point, we showed that hibris was induced by Notch (2-fold)
and repressed by Ras (10-fold), both by northern analysis and by in situ
hybridization in embryos (Artero et al.,
2001).
We used a combination of in situ hybridization, immunostaining and confocal microscopy to verify that the differential expression changes that we observed in these overexpression embryos reflected true differential expression in the wild-type situation. We analyzed the expression of nine genes from different functional categories in Table 1 (Figs 4, 6, and data not shown). For seven of these, we detected expression in the predicted type of myoblast. For two, ast and cadmus (see Materials and methods), we were not able to detect any specific staining in embryos by in situ hybridization. For those genes that fell into the category of 'specific role in muscle development uncertain', in situ hybridization of several (28%) showed expression in tissues other than somatic mesoderm that are present in the Toll10b background. These genes changed their expression levels in response to Ras or Notch, and may be Ras and Notch targets in non-mesodermal tissues.
We applied the most stringent test, mutational analysis, to a set of genes for which mutations are available. In addition to the three described in this paper, we have carried out preliminary analyses of another four FCM-enriched genes: EfTuM, Glutamine synthetase 1, cadmus and parcas. All four mutants have muscle defects, including muscle losses and aberrant muscle morphologies (see Materials and methods for details). Thus all the genes tested show some muscle defect, supporting the usefulness of our genetic and genomic approach.
Taken together, our data suggest that the majority of genes listed in Table 1 will play important roles in FCs or FCMs during muscle development. Some of these genes might not have been found in traditional forward genetic screens because of partial or complete genetic redundancy. Our data complement traditional forward genetic approaches for finding genes crucial for muscle morphogenesis.
Cell type-specific transcriptional regulators control FC and FCM
differentiation
Each of the thirty FCs per abdominal hemisegment is hypothesized to produce
its own unique combination of transcriptional regulators, though the evidence
for this is limited. In turn the combination of regulators would control the
morphology of the final muscle. Although several transcriptional regulators
have been linked to FC identity, the molecular description is far from
complete. Our screen contributed two more FC-specific genes. Previously known
markers, such as slouch or eve, once induced in the muscle
FC, are maintained throughout the remainder of development. Ubx,
which emerged from our screen, is a similarly simple case, as its transcripts
are steadily present in most FCs (Fig.
4 and data not shown). By contrast, we have identified more
complex patterns of gene expression in FCs, such as the transient
transcription of ase in a subset of FCs. The subsequent
transcriptional inactivation of ase may underlie temporal changes in
cell properties.
Even less is known about transcriptional regulators controlling FCM differentiation. Only one gene, lame duck, has been shown to have a role in FCMs. Our screen has uncovered three more potential players: dei, E(spl)mß and CG4136, confirming that FCMs follow their own, distinct, myogenic program. Discovering what aspects of FCM biology are controlled by these transcriptional regulators awaits analysis of the loss-of-function phenotypes.
FCs and FCMs each integrate Notch and Ras signaling pathways, but in
different ways
Notch and Ras signaling pathways interact during muscle progenitor
segregation (Carmena et al.,
2002). Our results suggest that phyl and
polychaetoid (pyd) may be additional links between the two
signaling pathways in FCs. phyl and pyd both interact
genetically with Notch and Delta (Chen et
al., 1996
; Pi et al.,
2001
). The transcription of phyl, which promotes neural
differentiation, is negatively regulated by Notch signaling during
specification of SOPs and their progeny
(Pi et al., 2001
). Our study
shows a similar regulation in muscle cells, where Notch signaling repressed
phyl expression and Ras signaling increased phyl expression
(Fig. 3). Likewise, in the
nervous system, the segregation of SOPs requires pyd, a Ras target
gene, to negatively regulate ac-sc complex expression. Similarly, Pyd
may restrict the muscle progenitor fate to a single cell, perhaps by
regulating lethal of scute transcription. Thus, Pyd would collaborate
with Notch signaling to restrict muscle progenitor fate to one cell.
FCMs appear to integrate Ras and Notch signaling differently. Two genes
whose transcripts were enriched under activated Notch conditions,
parcas and ast, have been implicated in Ras signaling in
other tissues, directly (ast)
(Kotarski et al., 1998) or
indirectly (parcas) (Yamadori et
al., 1999
; Schnorr et al.,
2001
). These data are suggestive of a role for Ras signaling in
the FCMs, in addition to its role in FC specification. In addition, Notch
signaling to FCMs may prime cells for subsequent Ras signaling during muscle
morphogenesis, much as occurs in FCs where Ras signaling primes the cell for
subsequent Notch signaling during asymmetric division of the muscle progenitor
(Carmena et al., 2002
).
Roles for ubiquination during muscle specification and
morphogenesis
Embryos that lack or ectopically express phyl have morphological
defects in specific muscles, for example, in LL1 and DO4 in response to
diminished phyl function, and in DT1 and LT4 in response to increased
phyl function. The morphological defects in the loss-of-function
embryos appear to be due to a failure to specify particular FCs, a conclusion
that is based upon missing or abnormal production of the FC marker Kr. In eye
development and SOP specification, Phyl directs degradation of the
transcriptional repressor Tramtrack
(Dickson, 1998). In a subset
of the primordial muscle cells, Phyl may work similarly, targeting Tramtrack
for degradation (Harrison and Travers,
1990
). The presence of Tramtrack would contribute to the specific
identity program of the muscle. As Tramtrack is expressed in the mesoderm
(Harrison and Travers, 1990
),
this possibility is likely. Alternatively, Phyl may be required for targeted
degradation of some other protein in a subset of FCs. The molecular partner
for Phyl during muscle differentiation is unknown, although preliminary data
suggest that sina is also expressed in somatic mesoderm and thus may
be its partner (K. Gonzalez and M.B., unpublished). Our studies have
identified a new role for Phyl in muscle progenitor specification and suggest
the importance of targeted ubiquitination for proper muscle patterning.
A role for ubiquitination in muscle differentiation is further reinforced
by the identification of the RING finger-containing protein Gol, induced by
activated Notch conditions, and CG17492, induced by activated Ras conditions.
Several RING-containing proteins function as E3 ubiquitin ligases, with the
ligase activity mapping to the RING motif itself
(Joazeiro and Weissman, 2000).
Ligase function has been experimentally confirmed for the Gol ortholog GREUL1
in Xenopus (Borchers et al.,
2002
). Thus, targeted protein degradation during muscle
morphogenesis could serve a host of crucial functions, such as protein
turnover, vesicle sorting, transcription factor activation and signal
degradation.
Muscle morphogenesis is controlled by both FCs and FCMs
The simplest view of the 'founder cell' hypothesis is that each FC contains
all the information for the development of a particular muscle. By contrast,
FCMs have been seen as a naïve group of myoblasts, entrained to a
particular muscle program upon fusion to the FC. Our work indicates that these
two groups of myoblasts have distinct transcriptional profiles. These data
raise the possibility of a greater role for FCMs in determining the final
morphology of the muscle and emphasize a need to characterize fully those FCM
genes listed in Table 1. For
example, our screen identified a protein kinase of the SR splice site selector
factors (SRPK) (Stojdl and Bell,
1999) whose transcripts are enriched in FCMs, suggesting that
regulation of the splicing machinery is important for muscle morphogenesis.
The Mhc gene undergoes spatially and temporally regulated alternative
splicing in body wall muscles conferring different physiological properties on
these muscles (Zhang and Bernstein,
2001
). This FCM-specific expression of SRPK may indicate that the
production of a particular Mhc isoform is regulated by the FCMs that
contribute to that muscle, rather than by the particular FC that seeds the
muscle. In addition, a number of observations suggest that FCMs may be a
diverse population of myoblasts, with different subsets having different
potential to contribute to the final muscle pattern. For example, hbs
expression suggests that only a subset of FCMs express the gene
(Artero et al., 2001
), and
twist expression in lame duck mutant embryos persists in a
subset of FCMs (Ruiz-Gomez et al., 2002). Our study provides additional genes
for exploring whether FCMs are a heterogeneous population of myoblasts as well
as determining the nature of FCM contribution to the final muscle.
The molecular events underlying complex morphological changes, such as migration, cell fusion, cell shape changes or changes in the physiology of a cell, require a rich and dynamic program of transcription changes. We have described approximately one-third of this transcriptional profile. The FC- or FCM-specific transcription of seven genes, and the mutant phenotype of four selected genes, allowed the definition of new muscle mutations that specifically affect the morphological traits of a subset of muscles. We now have the exciting prospect of exploring the functions of the numerous genes identified in this screen, and finding the molecular interactions among them that build perfectly organized muscles.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* Present address: Laboratory of Developmental Genetics, Department of
Genetics, University of Valencia, Dr Moliner 50, 46100 Burjasot, Spain
These authors contributed equally to this work
Present address: Developmental Biology Programme, European Molecular
Biology Laboratory, Heidelberg, Germany
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