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First published online 17 October 2007
doi: 10.1242/dev.008821
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1 Institute for Molecular Biology, University of Zürich, Winterthurerstr.
190, CH-8057 Zürich, Switzerland.
2 Skirball Institute of Biomolecular Medicine, New York University Medical
Center, 540 First Avenue, New York, NY 10016, USA.
Author for correspondence (e-mail:
noll{at}molbio.unizh.ch)
Accepted 21 August 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, Pox meso, Pax gene, lethal of scute conundrum, Somatic myogenesis, Muscle progenitors, Muscle patterning
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Baylies et al., 1998). Each of
the abdominal hemisegments A2-A7 has 30 identifiable individual muscles
(Bate, 1993) that develop from
the somatic mesoderm. This process is initiated when the invaginated mesoderm
migrates dorsolaterally under the ectoderm
(Beiman et al., 1996;
Gisselbrecht et al., 1996) and
is prepatterned by the segmentation genes
(Lee and Frasch, 2000;
Riechmann et al., 1997): the
product of sloppy paired (slp), whose activity is maintained
by the ectodermal Wingless (Wg) signal, restricts high levels of the bHLH
transcription factor Twist (Twi) to the mesodermal regions below the posterior
portions of the ectodermal parasegments
(Baylies et al., 1998). These
high levels of Twi function as a myogenic switch, separating the posterior
somatic and cardiac mesoderm from the anterior visceral mesoderm and fat body
(Baylies and Bate, 1996;
Dunin Borkowski et al., 1995).
When the dorsal migration of the mesoderm is complete, these metamerically
repeated Slp or high Twi domains are further subdivided along the dorsoventral
axis by the ectodermal signal Dpp
(Staehling-Hampton et al.,
1994). This signal restricts transcription of tinman
(tin) to the dorsal mesoderm, where its homeodomain protein specifies
heart and dorsal somatic mesoderm (Azpiazu
and Frasch, 1993; Bodmer,
1993; Bodmer et al.,
1990; Frasch,
1995). However, the determinant of the non-dorsal somatic mesoderm
remains largely unknown. It appears that Pox meso (Poxm) expression is
restricted to the ventral part of the high Twi domain by Dpp
(Staehling-Hampton et al.,
1994) to define the lateral and ventral somatic mesoderm anlage.
The characterization of the role of Poxm in somatic myogenesis is
therefore expected to fill an important gap in our understanding of the gene
network regulating this process.
Soon after this subdivision of the mesoderm, the proneural gene lethal
of scute [l(1)sc] begins to be expressed in at least 19
promuscular clusters of cells within the high Twi domain
(Carmena et al., 1995). From
these clusters, muscle progenitors are singled out by lateral inhibition
through Notch (N) and Ras signaling and are specified by the expression of
muscle-identity genes (Buff et al.,
1998; Carmena et al.,
1995; Carmena et al.,
1998a; Carmena et al.,
2002; Michelson et al.,
1998; Stathopoulos et al.,
2004). Cells not singled out begin to express the zinc finger
protein Lame duck (Lmd; also known as Minc), which specifies them as
fusion-competent myoblasts (FCMs) (Duan et
al., 2001; Ruiz-Gómez
et al., 2002). The progenitors divide to generate different muscle
founders, a muscle founder and an adult muscle precursor, or a founder and a
cell producing either two adult muscle precursors or two pericardial cells
(Carmena et al., 1995;
Carmena et al., 1998b;
Jagla et al., l998;
Nose et al., 1998;
Ruiz Gómez and Bate,
1997; Ruiz-Gómez et
al., 1997). Each founder forms an individual syncytial muscle
precursor by fusing with neighboring FCMs. One of the key steps in muscle
pattern formation is the specification of a muscle founder by the expression
of a specific set of muscle identity genes
(Bate, 1990;
Bour et al., 2000;
Dohrmann et al., 1990;
Ruiz-Gómez et al.,
2000; Rushton et al.,
1995). Although an increasing number of these genes have been
identified in recent years, the mechanisms that activate their transcription
are still poorly understood. Hence, it is important to identify the genes
whose products directly regulate the muscle identity genes.
In this study, we describe the functional characterization of the
Poxm gene. Poxm belongs to the Pax gene family whose members
encode transcription factors, including a paired domain
(Bopp et al., 1989) (reviewed
by Noll, 1993). The temporal
and spatial expression patterns of Poxm and its loss- and
gain-of-function phenotypes reported here demonstrate that it is required for
most ventral and lateral abdominal muscles to develop properly in all segments
and for the activation of muscle identity genes. In addition, Poxm
acts itself as muscle identity gene in a few muscles and thus plays a dual
role in somatic myogenesis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To produce transgenic UAS-Poxm lines, the 2.5 kb
full-length Poxm cDNA, P29c1, was inserted into the EcoRI
site of the pUAST vector (Brand and
Perrimon, 1993).
To generate transgenic um1-2-Poxm lines, an
XbaI-XhoI genomic fragment, extending from the upstream
XbaI site to the 5' leader
(Fig. 3E), and the adjacent 2.5
kb XhoI-PstI Poxm-cDNA fragment of P29c2, extending
from the leader to the 3' trailer beyond the first poly(A) addition site
(see Fig. S1A in the supplementary material), were cloned between the
XbaI and PstI sites of the P-element vector PW6
(Klemenz et al., 1987).
For germline transformation, these constructs, all verified by DNA
sequencing, were coinjected with the transposase carrying plasmid
P(
2-3) into w1118 or y w embryos. Three to
five independent lines of each construct were established and analyzed.
Immunohistochemistry and microscopy
To produce an anti-Poxm antiserum, a Poxm-cDNA fragment encoding
the 234 amino acids C-terminal to the paired-domain was cloned between the
BamHI and EcoRI sites of the pGEX-3X GST-fusion vector
(Pharmacia). The fusion protein was produced in bacteria, purified, and used
for immunization of rabbits as described previously
(Gutjahr et al., 1993a).
Antiserum was collected, affinity-purified, and used at a 1:10 dilution for
histochemical detection of Poxm as described
(Gutjahr et al., 1993a). The
purified anti-Poxm antiserum is free of any crossreactivity with embryonic
antigens as verified in homozygous PoxmR361 embryos.
The following primary antisera were also used: rabbit anti-MHC [myosin
heavy chain (Kiehart and Feghali,
1986)], rat anti-Slou (Carmena
et al., 1995), rabbit anti-Twi
(Roth et al., 1989), rat
anti-L(1)sc (provided by Ana Carmena, Instituto de Neurociencias, Alicante,
Spain), rabbit anti-ß-galactosidase (Cappel), rabbit anti-Tin
(Yin and Frasch, 1998), and
rabbit anti-GFP (Medical & Biological Laboratories, Nagoya, Japan).
Embryos were fixed and stained as described previously
(Gutjahr et al., 1993a).
Muscle patterns were visualized after staining with anti-MHC (or with anti-ß-galactosidase, when expressed under indirect control of Poxm) under bright-field microscopy by a Zeiss Axiophot. The fluorescent signals of double-labeled embryos were amplified by tyramide signal amplification (TSA; kits #12 and #25 from Invitrogen), and embryos were analyzed by a Leica SP1 confocal microscope.
Fly stocks
The following fly stocks were used. Oregon-R (Munich).
Df(3R)dsxd+R5/TM3, Sb
(Baker and Wolfner, 1988).
Df(3R)dsxM+R29/TM3, Sb
(Deák et al., 1997).
UAS-lacZ (Bloomington stock 1777). 24BGal4 (Bloomington
stock 1767). UAS-GFPnls (Bloomington stock 4775).
w*; Df(3R)159/TM3, Sb P{ry+;
hb-lacZ}. w1118; PoxmR361 red/TM3, Sb Ser
P{w+; hb-lacZ}. y w; Poxm8.4-Gal4/TM6B. y w;
Poxm1.8-Gal4 (2nd chromosome). y w; um1-2-Poxm;
PoxmR361 red/TM3, Sb P{ry+; hb-lacZ}.
UAS-Poxm (3rd chromosome). Df(1)sc19/FM7,
P{ry+; ftz-lacZ}. Df(1)sc19/FM7,
P{ry+; ftz-lacZ}; PoxmR361
red/TM3, Sb Ser P{w+; hb-lacZ}.
w*; l(3)S028206bS028206b19/TM3, Sb
(Deák et al., 1997).
w*; P{Mhc-tauGFP}/TM6B
(Chen et al., 2003).
P{PZ}rP298; ry506
(Nose et al., 1998).
w; lmd1/TM6B
(Duan et al., 2001).
Dmef222-21/CyO (Bour
et al., 1995). y w; Poxm1.8-lacZ (3rd
chromosome). y w; Poxm8.4-lacZ (3rd chromosome).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and was mapped to chromosomal band 84F11-12
(Bopp et al., 1989). It
extends over more than 20 kb that include two exons and many cis-regulatory
elements located in the upstream region and the large intron (see Fig. S1A in
the supplementary material) (Bopp et al.,
1989). The Poxm protein, predicted from the longest open reading
frame, consists of 370 amino acids and includes a paired domain close to its N
terminus and an octapeptide in its C-terminal moiety (see Fig. S1B in the
supplementary material) (Bopp et al.,
1989; Noll, 1993).
Except for its first 10 base pairs, the open reading frame is encoded entirely
by exon 2. The paired domain of Poxm belongs to the Pax1/9 class
(Bopp et al., 1989;
Noll, 1993) and displays 88%
identity and 92% similarity to mammalian Pax1/9-type paired domains
(Fu and Noll, 1997).
Expression of Poxm in the somatic mesoderm during myogenesis
In agreement with earlier results (Bopp
et al., 1989), Poxm protein is localized in the nucleus and first
detectable in the somatic mesoderm at early stage 10
(Fig. 1A,B). During stage 10,
Poxm becomes expressed in segmentally repeated mesodermal `stripes' underlying
the ectodermal parasegments 2-14, in the cephalic mesoderm, the proctodeal
anlage and a group of ectodermal cells in the clypeolabrum, which presumably
corresponds to part of the esophageal anlage
(Fig. 1C). At this stage, the
posterior boundaries of mesodermal Poxm coincide with those of ectodermal Gsb
(Bopp et al., 1989), which
largely coincide with the parasegmental borders
(Gutjahr et al., 1993b).
Consistent with these calibrations along the anteroposterior axis and those of
others (Riechmann et al.,
1997), we find that Poxm is expressed in cells of the high Twi
domain in the ventral and lateral mesoderm
(Fig. 2A-C). Since
Poxm is repressed in the dorsal portion of each segment by the
ectodermal signal Dpp (Staehling-Hampton
et al., 1994), the number of Poxm-expressing cells is reduced with
decreasing distance from the dorsal margin, thus forming a triangular pattern
(Fig. 1D). At this stage, Tin
expression is not yet completely restricted to the dorsal mesoderm
(Fig. 2D). Whereas high levels
of Tin in the dorsal region and Poxm are expressed in complementary patterns,
Poxm is coexpressed with low Tin levels in the ventral and lateral regions
(Fig. 2D-F). During stage 11
Poxm is restricted to fewer cells, some of which will form subsets of muscle
progenitors and cells of the promuscular clusters
(Fig. 1E,F), as evident from
its partial co-localization with L(1)sc
(Fig. 2G-I). During germ band
retraction, Poxm disappears from the most anterior mesodermal stripe and the
telson (Fig. 1E,G). By stage
12, Poxm expression is maintained only in six cells each of the abdominal
segments A1-A7 (Fig. 1H),
identified as founders of muscles DO3, DT1 and VA1-VA3, and as ventral adult
muscle precursor (VaP) by double-staining of Poxm and Slouch (Slou)
(Fig. 2J-L). At this time, it
becomes apparent that more cells express Poxm in the ventral regions of the
thoracic segments than of the abdominal segments
(Fig. 1G). In this study, we
focus on the role of Poxm in myogenesis of abdominal segments
A2-A7.
As myoblast fusion proceeds during stage 13, the number of Poxm-positive nuclei increases (Fig. 1I,J). These coincide with the precursors of muscles DT1 and VA1-3 (Fig. 1J,L-O), identified by double-staining of Poxm and MHC-tauGFP (Myosin heavy chain-tauGFP). During stage 15, Poxm expression begins to be reduced in the ventral clusters and is diminished in the dorsolateral region (Fig. 1K), from which it disappears during stage 16. By stage 17, Poxm is no longer detectable in the mesoderm or any of its derivatives. Outside the mesoderm, particularly striking is its expression in the developing esophagus and hindgut (Fig. 1A,C), where it is maintained at high levels throughout embryogenesis (Fig. 1E,G,I,K).
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Fate of Poxm-expressing cells during early and late myogenesis
To further analyze the nature and fate of Poxm-expressing cells during
early and late myogenesis, lacZ was expressed under the indirect
control of different Poxm upstream regions by the use of the Gal4/UAS
system (Brand and Perrimon,
1993). Because of the perdurance of ß-galactosidase
(ß-gal) resulting from (i) the amplification and delay of ß-gal
inherent in the Gal4/UAS system and (ii) the considerably enhanced stability
of both Gal4 and ß-gal proteins as compared to that of Poxm, we can
follow the fate of cells expressing Poxm during earlier embryonic stages by
examining ß-gal expression at later stages.
Under the control of a 1.8 kb upstream fragment of Poxm (Fig. 3E), ß-gal is expressed in a pattern similar, but not identical, to that of early Poxm in the mesoderm (Fig. 3A), presumably because of the temporal delay in expression of the Gal4/UAS system. A similar early expression pattern is observed (Fig. 3C) when lacZ is expressed under the control of an 8.4 kb upstream fragment (Fig. 3E). We have also examined ß-gal expression under the direct control of the 1.8 kb and 8.4 kb Poxm enhancers. In both cases, ß-gal and Poxm are coexpressed during early embryonic stages and no ectopic ß-gal is detectable (see Fig. S2 in the supplementary material).
Patterns of ß-gal expression were then examined at later stages in differentiating muscles. At stage 16, the 8.4 kb fragment supports strong lacZ expression in muscles DT1 and VA1-3 (Fig. 3D), in agreement with late Poxm expression, which is restricted to these muscles (Fig. 1N). In addition, however, muscles VL1-4, VO1-6, frequently LT3 and LT4, and occasionally muscle SBM are labeled by ß-gal, although at moderate or considerably lower intensities (Fig. 3D). By contrast, when lacZ is expressed under control of the 1.8 kb fragment, it is not detected in muscle DT1 and only at low or moderate levels in muscles VA1-3 (Fig. 3B). It follows that late Poxm expression is under the control of sequences present in the 8.4 kb but not the 1.8 kb fragment (Fig. 3E). Owing to perdurance, when expressed only under control of the early enhancer, ß-gal is also observed at moderate or low levels in the ventral muscles VL1-4, VO1-6, frequently in the lateral muscles LT3, LT4, LL1, LO1, SBM and rarely in LT2 and VT1 (Fig. 3B).
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) or Dmef222-21
(Bour et al., 1995) mutants,
in which fusion is blocked and muscle founders were marked by the
dumbfounded enhancer trap chromosome rP298-lacZ
(Nose et al., 1998). Because
of the perdurance of GFP, we can follow the fate of cells expressing early
Poxm by examining their expression of GFP at later stages. In
lmd1 embryos, GFP is expressed only in the ventral and
lateral portions of each segment at stage 15
(Fig. 3H). Since in the absence
of myoblast fusion most founders, marked by ß-gal
(Fig. 3I), also express GFP at
least weakly (Fig. 3J), we
conclude that cells expressing Poxm early during myogenesis will give rise to
most founders of the ventral and lateral muscles. In addition, Poxm is
expressed early in mesodermal cells that are not selected as progenitors, as
evident from the perdurance of GFP in many mesodermal cells different from
founders (Fig. 3J). Similar
results were obtained for Dmef222-21 mutants (not
shown).
Isolation and characterization of Poxm mutant alleles
The expression patterns of Poxm suggest that it plays a crucial
role in myogenesis. Assuming that absence of Poxm functions results
in lethality, we screened a collection of 1,400 lethal P-element insertions on
the third chromosome (Deák et al.,
1997) for lack of complementation with the deficiency
Df(3R)dsxD+R5 (see Fig. S1A
in the supplementary material) (Duncan and
Kaufman, 1975), which uncovers Poxm
(Bopp et al., 1989), and
subsequently for complementation with
Df(3R)dsxM+R29, whose
distal break point is located proximal to Poxm, at 84F6-7
(Baker et al., 1991). One
lethal insertion, P282, was identified that had inserted into the
neighboring gene, 5 kb downstream of the second exon of Poxm (see
Fig. S1A in the supplementary material). Embryos homozygous for P282
did not show any muscle defects. Imprecise excision of this P element
(Robertson et al., 1988)
produced a deficiency, Df(3R)159, whose distal breakpoint is located
about 10 kb upstream of the Poxm transcription start site (see Fig.
S1A in the supplementary material). Its proximal breakpoint maps distal to the
more proximal deficiency
Df(3R)dsxM+R29, with which
it complements. Embryos homozygous for Df(3R)159 show severe defects
in the larval somatic musculature.
Since Df(3R)159 deletes, in addition to Poxm, at least
another gene, the observed muscle phenotype might result from the absence of
more than just Poxm functions. Therefore, eight EMS-induced embryonic
lethal mutants, obtained in a screen for genes on the third chromosome
affecting neuromuscular connectivity (Sink
et al., 2001; Van Vactor et
al., 1993), that showed defects in muscle patterning were tested
for complementation with Df(3R)159. One of these mutants,
R361, failed to complement and showed the same larval muscle
phenotype as Df(3R)159, in homozygous and transheterozygous
conditions. No Poxm protein was detectable in either mutant (not shown).
Sequencing of R361 genomic DNA identified, in Poxm, a single
point mutation, PoxmR361, that converts a glutamine codon
at position 7 of the N-terminal paired domain into an amber stop codon and
hence is expected to result in a truncated N-terminal Poxm peptide of 14 amino
acids (see Fig. S1B in the supplementary material). It follows that
PoxmR361 is a null allele of Poxm.
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In the ventral region of Poxm mutant embryos, usually muscles VO4-6 are absent, whereas muscles VA1-3 are still present in most segments but are poorly developed, lacking their normal shape and attachment sites (Fig. 4G,H, Fig. 5A). Further analysis revealed that muscles VL3 and VL4 are frequently abnormal or missing, whereas muscles VL1 and VL2 are occasionally or rarely affected (Fig. 5A). Also muscles VO2 and VO1 are strongly and moderately disturbed, respectively (Fig. 5A).
In the dorsolateral region, muscle DT1, in most cases, is missing or
abnormal, whereas muscle DO3, which is derived from the same progenitor
(Carmena et al., 1995), is
mostly duplicated or abnormal and very rarely missing
(Fig. 4D,E,
Fig. 5A). Two additional
muscles, DA3 and DO4, are occasionally abnormal, whereas the two most
posterior lateral muscles, LO1 and LT4, are frequently missing and abnormal,
respectively (Fig. 5A). By
contrast, all dorsal muscles remain unaffected
(Fig. 4A,B,
Fig. 5A).
Ordering the muscles along the abcissa according to decreasing severity of
their Poxm mutant phenotype (red bars in
Fig. 5E) reveals a striking
correlation with the early triangular Poxm expression pattern
(Fig. 1D). Muscles located more
ventrally or more posteriorly in a segment are always more strongly affected
as compared to muscles located roughly at the same anteroposterior or
dorsoventral positions, respectively (Fig.
4N). For example, muscle VL4 is affected more severely than its
dorsal neighbor VL3, which is again more frequently abnormal than VL2 or VL1.
Similarly, the phenotype of muscle LT4 is stronger than that of its anterior
neighbors LT1-3. This phenotype suggests that it might be affected by a
function that depends on a dorsoventral as well as an anteroposterior
gradient, on which indeed the early Poxm expression pattern depends,
namely on Dpp (Staehling-Hampton et al.,
1994) and Wg (J.C. and M.N., unpublished), and which explains its
characteristic triangular shape (Fig.
1D).
Ectopic expression of Poxm in the mesoderm generates additional muscles
To test whether Poxm can determine muscle development, we expressed it
ectopically and analyzed its effect on myogenesis. 24BGal4 was used
to drive expression of UAS-Poxm in the entire mesoderm beginning at
mid stage 10 (Michelson,
1994). Ectopic Poxm produces a severely altered muscle pattern,
which varies among different segments and embryos. The most striking defects
occur in the dorsal and dorsolateral muscles, where Poxm is normally absent or
present at low levels (Fig.
4C,F). In the dorsal region, which includes four muscles in
wild-type embryos (Bate, 1993)
(Fig. 4A,J), ectopic muscles
are generated in most segments (Fig.
4C). Ectopic muscles similar in shape and orientation to muscle
DA3 occupy the dorsolateral region (Fig.
4C,F), which is largely free of muscles in wild-type embryos
(Fig. 4D). Usually several
muscles with abnormal shape occur at the position of muscle DT1
(Fig. 4F), whereas muscles LL1,
DO4 and DO5 exhibit aberrant shapes or are missing in some segments. In
addition, some of the lateral muscles are abnormally shaped. By contrast, the
ventral muscles, all of which exhibited a strong early Poxm expression
(Fig. 1C,D), remain largely
unaffected, although some muscle fibers appear enlarged
(Fig. 4I).
|
; Ruiz Gómez and
Bate, 1997; Ruiz-Gómez
et al., 1997), we suspected that Poxm also affects adult
muscle precursors. To test this hypothesis, we analyzed the effects of
loss-of-function and ectopic expression of Poxm on the expression of
Twi, which is present in all adult muscle precursors but not in larval
founders after germ band retraction (Bate
et al., 1991). In stage 14 wild-type embryos, adult muscle
precursors appear in four groups with a single precursor each in the ventral
and dorsal groups (VaP and DaP) and two each in the dorsolateral and lateral
groups (DLaPs and LaPs) (Fig.
6A) (Bate et al.,
1991; Ruiz Gómez and
Bate, 1997).
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In the dorsal region, after mesodermal ubiquitous expression of Poxm, on average two DaPs instead of one are present in about half of the segments (Fig. 6C,D). This result correlates with the appearance of ectopic dorsal muscles (Fig. 4C) and hence suggests that ectopic expression of Poxm leads to the production of supernumerary adult muscle precursors and muscle founders in the region where normally only a very low level of Poxm is expressed at early embryonic stages. In embryos lacking Poxm, however, DaPs remain largely unaffected (Fig. 6B,D).
In the ventral region, the number of VaPs is hardly changed not only in the presence of mesodermal ubiquitous Poxm but also in the absence of Poxm (Fig. 6B-D).
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;
Knirr et al., 1999;
Ruiz-Gómez et al.,
1997), occurs in a subset of muscle progenitors and their
offspring founders, some of which also express Poxm
(Fig. 1H). This raises the
possibility of an epistatic relationship between these genes. Early
Slou-expressing cells are arranged in three groups of muscle founders
(Fig. 7A): group I will
generate muscles LO1 and VT1; group II, muscles VA1-3 and the VaP; and group
III, muscles DO3 and DT1 (Carmena et al.,
1995; Dohrmann et al.,
1990). After stage 13, Slou remains expressed only in the
precursors of muscles DT1, VT1 and VA2
(Carmena et al., 1995;
Dohrmann et al., 1990)
(Fig. 7C,E), two of which, DT1
and VA2, also express Poxm (Fig.
1J,L). In PoxmR361 embryos, Slou protein is
expressed in groups I and II, yet is absent from group III in most, though
occasionally observed in more posterior, abdominal segments during late stage
12 (Fig. 7B). After stage 13,
Slou is detectable only in the precursor of muscle VT1 but no longer
maintained in that of VA2 in abdominal segments
(Fig. 7D). Therefore, Poxm is
essential for the activation of slou in the progenitor of muscle DT1
and for its maintenance in the precursor of muscle VA2. In 24BGal4/UAS-Poxm embryos, in which Poxm is ubiquitously expressed in the mesoderm, additional muscles expressing Slou were found in the dorsolateral portion of some segments (Fig. 7F), which suggests that in these cells ectopic Poxm suffices to activate slou and corroborates the observation that Poxm acts upstream of the muscle identity gene slou.
Early Poxm largely rescues the muscle phenotype of Poxm mutants
Since Poxm is expressed during early myogenesis in cells that later give
rise to progenitors of most of the ventral and lateral muscles, it may play an
important role in the initiation of muscle patterning. To investigate which
part of the PoxmR361 muscle phenotype results from the
loss of this early Poxm function, a transgene expressing
Poxm only during the early myogenic stages
(Fig. 3F,G),
um1-2-Poxm, was introduced into PoxmR361 embryos.
In these embryos, the phenotypes of muscles VO4-6, VL2-VL4, VO2, VO1, LO1, LT4
and VT1 are efficiently rescued (Fig.
4K; Fig. 5B,E). The
only muscles affected in Poxm mutants
(Fig. 5A) that are only
slightly rescued by early Poxm (Fig.
5B) are DT1, DO3 and VA1-3, in which Poxm is also expressed during
later stages in their founders and/or muscle precursors
(Fig. 4K;
Fig. 5B,E). These results
strongly suggest that Poxm exerts an early function, demarcating a
mesodermal domain of competence for ventral, lateral and dorsolateral somatic
muscle development.
Partial redundancy of early Poxm and l(1)sc functions in somatic myogenesis
The partial penetrance of the Poxm muscle phenotype
(Fig. 5A) suggests that the
early Poxm function is largely redundant with that of other genes, an
argument also raised to explain the weak muscle phenotype of l(1)sc
mutants (Carmena et al.,
1995). The l(1)sc gene encodes a bHLH transcription
factor the function of which is thought to be required for the selection of
muscle progenitors (Baylies et al.,
1998; Carmena et al.,
1995). Therefore, we examined the effect of Poxm and
l(1)sc mutations on larval muscle development in single and double
mutant embryos (Fig.
5A,C,D).
In agreement with earlier studies
(Carmena et al., 1995),
l(1)sc mutants exhibit a weak muscle phenotype, which deviates only
slightly from that of wild-type embryos
(Fig. 4L,
Fig. 5C). Although
PoxmR361 embryos show a considerably stronger muscle
phenotype, most lateral and dorsal muscles are normal
(Fig. 5A). Assuming that
Poxm and l(1)sc act independently in muscle development, we
expect that the probability of a muscle being wild-type in Df(1)
l(1)sc19/Y; PoxmR361 embryos is the
product of the probabilities of the muscle being wild-type in the single
mutants. Conversely, if we find significantly enhanced probabilities for
muscle defects in double mutants, we may conclude that Poxm and
l(1)sc exhibit partially redundant functions during muscle
development. Applying this test to the results summarized in
Fig. 5A,C,D, we find that most
muscles are affected independently or nearly independently, with some notable
exceptions. These concern muscles VL1-3, SBM, VO1, VO2, DT1, LT3, LT4 and VA3
that are more often absent. Some muscles are strongly affected in
Poxm null mutants, such as muscles VO4-6 or muscles VA1-3. Among the
other muscles, the more ventral and the more posterior a muscle is located
within a segment, the more probable it is that it will show an enhanced
phenotype in double mutants (Fig.
5E). Clearly, there is some redundancy between Poxm and
l(1)sc functions in the somatic mesoderm, which is restricted largely
to ventral and posterior muscles.
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; Ruiz-Gómez et
al., 1997). Thus, late expression of Poxm in the
precursor of muscle DT1, but not of DO3, is crucial for their distinction and
hence serves a muscle identity function. However, a more detailed analysis
shows that muscle DT1 is missing in only two thirds (23/34) of all cases in
which muscle DO3 is duplicated (see Table S1 in the supplementary material).
In the remaining 11 cases, muscle DT1 is normal (4), abnormal (6) or
duplicated (1). This finding suggests that the late Poxm function is
necessary in about 10% (11/108) of all cases to prevent an additional division
that generates a second founder of muscle DO3. Absence of Poxm in their founders results in abnormal muscles VA1-3 (Fig. 5A) that cannot be rescued by the early Poxm function (Fig. 5B), which suggests that their proper specification also depends on the late function of Poxm.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Michelson et al.,
1998; Yin and Frasch,
1998). Poxm and tin thus subdivide the posterior
Slp domain into ventral and dorsal subdomains in a manner similar to the
partitioning by serpent and bap of the anterior Eve domain
into the ventral fat body and the dorsal visceral mesoderm anlagen
(Azpiazu et al., 1996;
Riechmann et al., 1998). After
selection of muscle progenitors, proper development of a few muscles still
depends on Poxm, which is expressed in muscles DT1 and VA1-3. This
late function of Poxm participates in founder specification and
muscle differentiation, as is characteristic for muscle identity genes.
Finally, our findings suggest a solution to a conceptual problem of the
current model of somatic myogenesis, the l(1)sc conundrum.
Early Poxm specifies competence for somatic myogenesis in partial redundancy with similar functions of L(1)sc
The muscle phenotype of Poxm mutant embryos and its rescue by
early Poxm expression shows that the early Poxm function is crucial
for the proper development of many ventral and lateral muscles
(Fig. 5A,B). In addition, the
generation of ectopic dorsal and dorsolateral muscles by ectopic Poxm suggests
that Poxm has the ability to change cell fates and render cells
competent for myogenesis. Therefore, we propose that early Poxm demarcates a
ventral and lateral domain of competence for somatic myogenesis.
The partial penetrance of the Poxm mutant phenotype implies the
existence of other competence domain genes performing partially redundant
functions. We have shown that Poxm and L(1)sc partially co-localize in the
promuscular clusters and muscle progenitors
(Fig. 2G-I). In addition, a
detailed analysis of l(1)sc and Poxm single and double
mutants demonstrates that their functions are partially redundant
(Fig. 5A,C,D). Since the muscle
phenotype of l(1)sc; Poxm double mutants still shows partial
penetrance (Fig. 5D),
additional competence domain genes should be expressed in the Slp domain. One
of them is probably tin, which is initially expressed in the entire
early mesoderm (Azpiazu and Frasch,
1993; Bodmer,
1993; Bodmer et al.,
1990), because tin mutants affect muscle development in
the dorsal as well as lateral and ventral Slp domain
(Azpiazu and Frasch, 1993;
Michelson et al., 1998).
Another candidate is D-six4, which is required for the development of
specific muscles that arise from the dorsolateral and ventral regions
(Clark et al., 2006).
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; Halfon et
al., 2000). These competence domain genes act in a cooperative
manner to determine the identities of specific muscles by regulating the
expression of the muscle identity genes. When one of them is inactivated, in
some cells active competence domain genes can partially compensate for the
inactive gene by activating its target genes such that these sometimes, but
not always, exceed the threshold levels required for normal development.
Hence, muscles derived from these cells exhibit a mutant phenotype with
partial penetrance. For other cells, active competence domain genes can
compensate completely for the missing gene activity such that these cells will
adopt the proper fate and the muscles develop normally. This illustrates that
competence is not a matter of `all' or `nothing' for muscle development. The
deeper reason for this, we deem, is that the genetic program regulating
myogenesis is not organized in a hierarchical fashion but rather as a complex
gene network (Fig. 8) that has
an integrated function which is much more stable against mutations within the
network than a hierarchical regulation would be.
Poxm is a muscle identity gene activating the muscle identity gene slou
Muscle identity genes usually encode transcription factors, such as Slou,
Nau, Ap, Vg, Kr, Eve, Msh, Lb, Run and Kn
(Bate et al., 1993;
Bourgouin et al., 1992;
Carmena et al., 2002;
Dohrmann et al., 1990;
Frasch et al., 1987;
Jagla et al., 2002;
Michelson et al., 1990;
Knirr et al., 1999;
Nose et al., 1998;
Ruiz Gómez and Bate,
1997; Ruiz-Gómez et
al., 1997), that are expressed in subsets of muscle progenitors
and founders and determine in a combinatorial fashion the identity of each
muscle founder and its subsequent differentiation into a specific muscle of
defined size, shape, attachment sites, and innervation
(Baylies et al., 1998;
Dohrmann et al., 1990;
Ruiz-Gómez et al.,
1997). We envision the activation of these genes in promuscular
clusters or, after lateral inhibition, in muscle progenitors
(Carmena et al., 1995;
Carmena et al., 1998a) by Twi
and/or the products of competence domain genes and through combinations of
localized extracellular signals from the ectoderm and mesoderm
(Azpiazu and Frasch, 1993;
Halfon et al., 2000). During
asymmetric division of progenitors, expression of a muscle identity gene may
be maintained in one or both of the two sibling founders, or it may persist in
the founder when division generates a founder and an adult muscle precursor.
Late expression of Poxm illustrates all three cases. It is expressed in
progenitors P26/27 and P29/VaP, which are derived from promuscular cluster 10
and give rise to the founders of muscles VA1 (F26) and VA2 (F27), and to the
founder of muscle VA3 (F29) and the ventral adult precursor VaP
(Carmena et al., 1995;
Dohrmann et al., 1990). Poxm
is also expressed in the progenitor derived from cluster 13, P11/18, which
generates the founders of muscles DO3 (F11) and DT1 (F18). Although Poxm
expression persists in F29 and F18 but not in their siblings, it is maintained
in both sibling founders F26 and F27.
The late function of Poxm is identified as a muscle identity function by the high correlation between absence of muscle DT1 and corresponding duplication of muscle DO3 in Poxm mutants (Fig. 5A and see Table S1 in the supplementary material). If Poxm was the sole determinant discriminating between F11 and F18, mesodermal ubiquitous expression of Poxm would be expected to transform muscle DO3 into DT1. Our results confirm the presence of additional muscles in the region of muscle DT1. It is possible that one of these originates from a transformed F11, but it is impossible to tell whether muscle DO3 is missing (Fig. 4F) because additional muscles have been recruited.
It has been shown that in the process of muscle diversification, identity
genes may repress or activate other identity genes in progenitors and founders
(Jagla et al., 1998;
Jagla et al., 2002;
Knirr et al., 1999;
Nose et al., 1998;
Ruiz-Gómez et al.,
1997). We found that the muscle identity gene slou fails
to be activated in P11/18 of Poxm mutants. The simplest explanation
of this result is that activation and maintenance of slou expression
depend on Poxm in P11/18 and its offspring founders. In addition,
slou expression is not maintained in F27 of Poxm mutants
despite its initial activation in P26/27. It therefore appears that in P26/27
and its offspring F26 and F27, in addition to Kruppel (Kr)
(Ruiz-Gómez et al.,
1997), Poxm is necessary for the maintenance of slou
expression. Although Poxm expression is maintained in both F26 and F27,
slou expression is restricted to F27 because Kr is repressed
in F26 by N signaling. Apparently, Kr is the crucial determinant that
distinguishes F26 from F27, as F27 is altered to F26 in Kr or
numb mutants (Ruiz Gómez
and Bate, 1997;
Ruiz-Gómez et al.,
1997).
As Poxm is expressed in both F26 and F27, whereas its expression
is restricted to F18 and not maintained in F11, its late expression in F26 and
F27 must be regulated differently from that in F11 and F18 where it appears to
be subject to asymmetric N signaling (Ruiz
Gómez and Bate, 1997) repressing Poxm in F11.
These considerations imply that slou is part of the same gene
network as Poxm, a conclusion consistent with our gene network
hypothesis since, in the first test of this hypothesis, slou had been
isolated as a PRD 9 gene on the basis of its homology to the prd gene
(Frigerio et al., 1986).
A solution of the l(1)sc conundrum
The mechanism of progenitor selection from the somatic mesoderm depends on
a process of lateral inhibition very similar to that of neuroblast or sensory
organ precursor (SOP) selection in the neuroectoderm from proneural clusters
expressing the proneural genes (Bate et
al., 1993; Corbin et al.,
1991). Because of this similarity, a search among proneural genes
for `promuscular' genes expressed in the somatic mesoderm was performed
(Carmena et al., 1995). This
search identified a single proneural gene, l(1)sc, a member of the
achaete-scute complex (AS-C), that is expressed in promuscular
clusters of the somatic mesoderm. It was, therefore, attractive to consider
its function in myogenesis to be analogous to that of proneural genes in
neurogenesis (Carmena et al.,
1995; Carmena et al.,
1998a). However, whereas proneural genes confer on neuroectodermal
cells the ability to become neural precursors rather than epidermal cells,
which is their default fate (Campuzano and
Modolell, 1992), l(1)sc does not seem to confer on
mesodermal cells the ability to undergo somatic myogenesis instead of becoming
part of the visceral mesoderm, heart or fat body. When L(1)sc was expressed in
the entire mesoderm from stage 8 onward, other mesodermal tissues could not be
transformed into somatic mesoderm (Carmena
et al., 1995), whereas a deficiency of l(1)sc resulted in
only minor defects of somatic muscle development
(Fig. 5C)
(Carmena et al., 1995). In
addition, as the l(1)sc muscle mutant phenotype can be rescued by
ubiquitous mesodermal L(1)sc expression
(Carmena et al., 1995), its
expression in clusters is not decisive for the formation of promuscular
clusters and, therefore, l(1)sc cannot play the decisive role in the
development of larval body wall muscles that has been proposed
(Carmena et al., 1995). Thus,
although l(1)sc serves as an excellent marker for promuscular
clusters, it lacks a property expected to be crucial for a promuscular gene.
Are there genes that might qualify as promuscular genes and thus extend the
close evolutionary relationship of progenitor selection between myogenesis and
neurogenesis (Jan and Jan,
1993)?
There is indeed a gene that is homologous to proneural genes and expressed
in the somatic mesoderm, in the absence of which somatic myogenesis is
seriously disturbed. This gene is twi, whose function at stages 10
and 11 more closely corresponds to that of a promuscular gene and which, like
l(1)sc, encodes a bHLH transcription factor. Although Twi is also
expressed earlier when it is required for mesoderm specification during
gastrulation, this early function can be distinguished from its later
`promuscular' function in temperature-sensitive mutants
(Baylies and Bate, 1996). In
these mutants, only high levels of Twi activity, necessary for the formation
of the somatic mesoderm, are abolished and no normal somatic muscles develop
(Baylies and Bate, 1996).
Moreover, ubiquitous expression of high levels of Twi in the mesoderm blocks
all other mesodermal fates, transforming them to somatic mesoderm
(Castanon et al., 2001). Since
the subsequent patterning of somatic muscles depends critically on the
relative levels of the products of twi and the proneural gene
da (Castanon et al.,
2001), it seems appropriate to consider them both as promuscular
genes.
In addition to its strict requirement for somatic myogenesis, the proposed
promuscular function of twi may be subject to lateral inhibition by N
signaling, in further analogy to proneural functions in neurogenesis. This is
apparent from experiments demonstrating that the restriction of high Twi
levels to the Slp domain during stage 9 depends on N signaling
(Brennan et al., 1999;
Tapanes-Castillo and Baylies,
2004), which downregulates twi in the mesoderm underlying
the anterior regions of parasegments where Slp does not override it
(Riechmann et al., 1997).
Since this process acts directly on an identified twi enhancer during
stages 9 and 10 (Tapanes-Castillo and
Baylies, 2004), it is conceivable that this enhancer also responds
to N signaling during the subsequent lateral inhibition. An alternative,
though not mutually exclusive, mechanism for the downregulation of
twi implicates the Gli-related zinc finger transcription factor Lmd
(Minc), whose expression is maintained by N signaling and in the absence of
which twi is not downregulated in fusion-competent myoblasts
(Duan et al., 2001;
Ruiz-Gómez et al.,
2002).
During lateral inhibition, loss of Twi precedes that of L(1)sc in the
promuscular clusters (Carmena et al.,
1995). It is therefore possible that l(1)sc expression in
these cells also depends on high levels of Twi, i.e. on Twi homodimers
(Fig. 8). Consistent with this
interpretation, shifting the equilibrium between Twi homodimers and Twi-Da
heterodimers in favor of the latter represses l(1)sc
(Castanon et al., 2001). Since
early Poxm expression also depends on Twi (J.C. and M.N., unpublished),
Poxm would be similarly repressed in promuscular clusters through
lateral inhibition, either indirectly by repression of twi and/or
directly by Twi/Da heterodimers. Such a mechanism might apply generally to
both competence domain genes and muscle identity genes during lateral
inhibition of promuscular clusters.
Thus, twi satisfies two criteria considered to be crucial for a
promuscular gene in analogy to those of proneural genes in neurogenesis.
However, a third criterion is not fulfilled by twi: its expression,
in contrast to that of proneural genes in the neuroectoderm, is ubiquitous
rather than restricted to promuscular clusters although this criterion is not
a crucial property of proneural genes
(Rodríguez et al.,
1990). Yet promuscular clusters from which the myogenic
progenitors are selected exist, as evident from the pattern of l(1)sc
expression (Carmena et al.,
1995). These promuscular clusters depend on combinations of the
long-range ectodermal signals Wg and Dpp
(Lee and Frasch, 2000;
Carmena et al., 1998a) and the
localized activities of the EGF signal Spi in the mesoderm and the FGF signals
Pyr and Ths in the ectoderm (Buff et al.,
1998; Carmena et al.,
1998a
