1 Philipps-Universität Marburg, Fachbereich Biologie,
Zoologie/Entwicklungsbiologie, Karl-von-Frisch-Strasse, 35039 Marburg,
Germany
2 Institut für Neuro- und Verhaltensbiologie, Westfälische
Wilhelms-Universität Münster, Badestrasse 9, 48149 Münster,
Germany
3 Umeå Center for Molecular Pathogenesis, Building 6L, Umeå
University, S-90187, Sweden
4 Institut für Allgemeine und Spezielle Zoologie, Allgemeine Zoologie und
Entwicklungsbiologie, Justus-Liebig-Universität Gießen,
Stephanstraße 24, 35390 Gießen, Germany
Author for correspondence (e-mail:
anne.holz{at}allzool.bio.uni-giessen.de)
Accepted 7 November 2003
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SUMMARY |
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Searching for genes involved in the determination and differentiation of the different visceral cell types, we identified two independent mutations causing loss of visceral midgut muscles. In both of these mutants visceral muscle founder cells are missing and the visceral mesoderm consists of fusion-competent myoblasts only. Thus, no fusion occurs resulting in a complete disruption of visceral myogenesis. Subsequent characterisation of the mutations revealed that they are novel alleles of jelly belly (jeb) and the Drosophila Alk homologue named milliways (miliAlk). We show that the process of founder cell determination in the visceral mesoderm depends on Jeb signalling via the Milliways/Alk receptor.
Moreover, we demonstrate that in the somatic mesoderm determination of the opposite cell type, the fusion-competent myoblasts, also depends on Jeb and Alk, revealing different roles for Jeb signalling in specifying myoblast diversity. This novel mechanism uncovers a crosstalk between somatic and visceral mesoderm leading not only to the determination of different cell types but also maintains the separation of mesodermal tissues, the somatic and splanchnic mesoderm.
Key words: Drosophila, Myogenesis, Visceral and Somatic muscles, Founder cells, Fusion-competent cells, Myoblast fusion, Notch, Delta, lethal of scute, jelly belly, Alk, RTK signalling
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Introduction |
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Recently, Weiss et al. (Weiss et al.,
2001) identified a novel signalling process that induces visceral
cell identities. The jelly belly (jeb) gene encodes a
secreted protein that is produced from somatic mesodermal cells and taken up
by visceral cells. In jeb mutants, no visceral muscles develop due to
a failure of differentiation in the existing Bap-expressing visceral
precursors. The emerging picture shows, on the one hand, visceral
determination genes such as tin, bap and bin, which are
responsible for subdividing the mesoderm into different tissues and, on the
other hand, inductive signals like Jeb, which promote interactions between
these tissues.
The differentiated visceral musculature of the Drosophila midgut
consists of an inner layer of circular muscles and an outer layer of
longitudinal muscles (Campos-Ortega and
Hartenstein, 1997), the latter persisting throughout metamorphosis
(Klapper, 2000
). The founder
cells of the longitudinal muscles have a distinct primordium at the
posteriormost part of the mesoderm (Tepass
and Hartenstein, 1994
;
Georgias et al., 1997
;
Kusch and Reuter, 1999
),
whereas the circular visceral founder cells and all fusion-competent myoblasts
(fcms) for both visceral muscle types originate from the trunk mesoderm
(Klapper et al., 2002
).
Recently, it has been shown that both types of visceral muscles are syncytial
and arise by fusion of founders and fcms
(Klapper et al., 2001
;
San Martin et al., 2001
;
Klapper et al., 2002
). These
two classes of myoblasts are closely associated in a band of visceral
precursors, which express markers such as Fasciclin III
(Patel et al., 1987
) and are
characterised by binou expression
(Zaffran et al., 2001
). The
ventralmost row of these visceral myoblasts consists of dumbfounded/kin of
irre (duf/kirre)-expressing founder cells with a characteristic
columnar shape. The more dorsally located fcms are characterised as such by a
more globular morphology and by expression of sticks and stones
(sns). During stage 12, binucleate circular muscles are built via
fusion of these founders and fcms. This fusion process is disturbed in
myoblast city (mbc), duf/kirre and sns
mutants, which are also known to be defective in somatic muscle fusion,
indicating that the founder cell hypothesis applies both to somatic and
visceral myogenesis. Thus, several of the known genetic components are common
between somatic as well as visceral myoblast fusion
(Klapper et al., 2001
;
San Martin et al., 2001
;
Klapper et al., 2002
).
Differentiation of the syncytial somatic muscles depends on the
determination of founder and fusion-competent myoblasts
(Bate, 1990;
Dohrmann et al., 1990
). These
two classes of myoblasts are specified by lateral inhibition by the neurogenic
genes Notch and Delta from a group of equivalent somatic
mesodermal cells (Carmena et al.,
2002
). As a consequence, expression of the proneural gene
lethal of scute (l'sc) is restricted to muscle progenitor
cells. These progenitors divide asymmetrically and give rise to muscle founder
cells (Carmena et al., 1995
),
which are characterised by differential expression of myogenic genes (reviewed
by Baylies et al., 1998
;
Paululat et al., 1999
). After
establishment of myoblast diversity, the fusion process starts, leading in a
first fusion step to muscle precursor cells and in a second fusion step to
formation of mature myotubes (Doberstein
et al., 1997
; Rau et al.,
2001
). This process can be disturbed at distinct levels (reviewed
by Dworak and Sink, 2002
;
Taylor, 2002
). Besides lateral
inhibition and determination through distinct transcription factors cell-cell
signalling is an important mechanism in myogenesis.
Receptor tyrosine kinases (RTKs) are involved in intercellular
communication in a wide range of processes. RTKs are composed of three
domains: an extracellular ligand-binding domain, a single membrane-spanning
domain and a cytoplasmic catalytic domain
(Yarden and Ullrich, 1988).
Ligand binding to the extracellular domain induces activation of the kinase on
the cytoplasmic side, which initiates the intracellular signalling. The
activated RTKs phosphorylate themselves and cytoplasmic substrates, leading to
activation of a number of downstream signalling molecules, and ultimately
induce changes in gene expression and the phenotypic state of the cell
(Fantl et al., 1993
;
van der Geer et al., 1994
).
RTKs thus play important roles in cellular proliferation and differentiation.
The role of RTKs during embryonic development and especially during the
determination of distinct cell types has been studied in detail in
Drosophila (reviewed by Rebay,
2002
). During specification of muscle progenitor cells from
equivalent cell clusters, the Ras/MAPK pathway functions as an inductive
cellular determination signal. This pathway is activated by both epidermal and
fibroblast growth factor receptors in the dorsal embryonic mesoderm
[(Gabay et al., 1997
); DER
(Buff et al., 1998
) and
htl (Michelson et al.,
1998
)], while Notch antagonises this activity by lateral
inhibition (Carmena et al.,
2002
). Recently, a novel RTK named DAlk (Drosophila
Anaplastic Lymphoma Kinase; Alk - FlyBase) was described which is expressed
specifically in the developing visceral mesoderm and CNS of
Drosophila (Lorén et al.,
2001
). Furthermore, activation of Alk mediated signalling is
required for embryonic gut development and more specifically for the
activation of MAP kinase in the visceral mesoderm
(Lorén et al., 2003
).
As Alk drives MAPK activation in the visceral mesoderm it is an obvious
candidate to be involved in determination of distinct visceral cell types.
In a search for genes involved in the determination of visceral cell types,
we screened a collection of EMS-induced lethal mutations established
previously (Hummel et al.,
1999a; Hummel et al.,
1999b
) and identified two independent mutations with a nearly
identical phenotype. Both exhibit a loss of circular visceral founder cells at
early stages of visceral development, whereas fusion-competent visceral
myoblasts are specified correctly and express markers like Fas3 and Sns. This
results in a complete absence of visceral midgut muscles. Complementation
analysis revealed that one mutation belongs to the previously described
jelly belly (jeb) gene, while the second mutation named
milliways (miliAlk) represents a Alk
allele. We describe the crucial role of these genes in the process of visceral
founder cell determination and analyse the role of Notch and
Delta during the distinction between visceral cell types. Moreover,
we uncover Jeb signalling via the Alk RTK as a new determination step for the
fusion-competent cells of the somatic mesoderm.
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Materials and methods |
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The collection of EMS mutagenised flies was obtained from Christian
Klämbt (Hummel et al.,
1999a; Hummel et al.,
1999b
). To screen for mutants with defects in the determination of
founder cells in the visceral mesoderm, we stained 180 lines on the second and
270 lines on the third chromosome, with anti-Fas 3 antibodies
(Patel et al., 1987
). We
isolated a new jeb allele, jebweli and a
new Alk allele, which we named miliAlk.
These two mutants were used for all experiments described herein.
For complementation tests we used jebk05644
(Weiss et al., 2001) from the
Bloomington Drosophila Stock Center, and
Df(2R)Alk
21 which is a deficiency covering 53C
(Lorén et al., 2003
).
ß-galactosidase-expression in founder cells was achieved using the
enhancer trap line rP298-lacZ
(Nose et al., 1998
;
Klapper et al., 2002
) and in
the entire mesoderm using bap-lacZ
(Azpiazu and Frasch, 1993
;
Zaffran et al., 2001
).
Overexpression studies were carried out using a bap-GAL4 driver line
(Zaffran et al., 2001
), which
drives expression in the circular visceral mesoderm from stage 10 onwards or a
twist-GAL4 driverline (SG24-GAL4), which drives expression
in the entire mesoderm (gift from A. Michelson). As UAS lines we used
UAS-Alk (Lorén et al.,
2001
) and UAS-jeb flies
(Weiss et al., 2001
). All
overexpression studies were carried out at 25°C.
Immunohistochemical staining
Immunostaining was performed as described previously
(Knirr et al., 1999;
Klapper et al., 2002
). The
mouse Fas3 antiserum (Patel et al.,
1987
) was used for the visualisation of the visceral mesoderm
cells (diluted 1:5, gift from C. Klämbt). A rabbit
anti-ß-galactosidase antibody (Biotrend, diluted 1:2500) was used to
visualise muscle founder cells in the enhancer trap line rP298-lacZ
and a mouse anti-ß-galactosidase antibody (Promega, diluted 1:500) was
used to visualise bap-lacZ expression. For analysis of the somatic
mesoderm, we used rabbit anti-ß3tubulin antibodies at a dilution of
1:2500 (Leiss et al., 1988
).
The rabbit anti-Alk (diluted 1:500)
(Lorén et al., 2003
),
rabbit anti-Jeb (diluted 1:100) (Weiss et
al., 2001
), rabbit anti-Lmd (diluted 1:1000)
(Duan et al., 2001
), mouse
anti-Notch (diluted 1:10) (Fuß and
Hoch, 2002
) and mouse anti-Delta antiserum (diluted 1:50,
Developmental Studies Hybridoma Bank) were used in combination with the TSA
signal amplification kit (NEN). For secondary antibodies, we used Cy2- and
Cy3-labelled antibodies made in goat against rabbit and mouse from Dianova
(diluted 1:40 and 1:100). The embryos were embedded in Fluoromount G (Southern
Biotechnology Associates) and photos were taken under Nomarski optics with a
Zeiss Axiophot microscope or a Leitz confocal microscope and processed with
Adobe Photoshop 6.0 (Adobe Systems).
In situ hybridisation
In order to visualise fcms in the mesoderm, whole-mount fluorescent DNA
hybridisation was performed with random digoxigenin-labelled sticks and
stones cDNA probes according to Knirr et al.
(Knirr et al., 1999). The
sns cDNA was a gift from S. Abmayr
(Bour et al., 2000
). For the
combination with antibody stainings to visualise ß-galactosidase
expression we used anti-ß-galactosidase antiserum (Cappel) at a dilution
of 1:3000.
Lethal phase analysis
To test the lethality of the progeny from UAS-GAL4 crosses we
mated homozygous lines carrying the founder cell marker rP298-lacZ,
collected eggs for 24 hours and allowed further development for another 48
hours at 25°C. Afterwards, we counted at least 1000 progeny of each
cross.
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Results and discussion |
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Notch and Delta play important roles in various developmental processes and mutations in either gene lead to strong developmental defects during embryogenesis. Thus, it is difficult to analyse whether the visible defects in the visceral mesoderm are due to defects in the determination of the founder cells or are a result of secondary effects. In Notch mutant embryos more founder cells appear to be present in the visceral mesoderm (Fig. 1A,B). The visceral fcms seem to be reduced compared with the wild-type expression of sticks and stones (sns) as a marker for these cells (Fig. 1D,E). This reduction is not as severe as in the somatic mesoderm but still quite obvious. In Delta mutants, the number of founder cells also seems to be increased in comparison with the wild type and the fcms are reduced in mutant embryos (Fig. 1C,F).
|
To exclude the possibility that the described defects are due to
non-endogenous effects induced by the overexpression of the examined genes in
the wrong tissue, we analysed wild-type Notch expression and found that it is
indeed expressed in the visceral mesoderm. Notch is localised at cell
membranes in the entire visceral mesoderm during stage 11, with expression
becoming weaker in the fcms of the visceral mesoderm, which continue to
express bap-lacZ after the determination process is finished
(Fig. 1G,H, arrowhead in G).
This reduction of Notch expression in the fcms after the establishment of the
founder cells is similar to its expression in the somatic mesoderm, where
Notch expression is also highest in the progenitor cell after the
determination process is completed
(Carmena et al., 2002).
Surprisingly, the analysis of Delta expression exhibits that this Notch ligand
is not expressed in the visceral mesoderm during founder cell formation. Delta
expression was found in adjacent, probably somatic cells
(Fig. 1I,J) and might be needed
there to participate in the visceral determination process, as indicated by
the increased number of founder cells and reduced number of fcms in
Delta mutants. Even though Dl is expressed in the cells surrounding
the visceral mesoderm, ectopic expression of UAS-Dl in these cells
with a twi-GAL4 driver line does not result in an obvious phenotype
(data not shown), which might be due to the fact that the amount of Delta in
this tissue is not the limiting factor that restricts Notch
signalling. Another explanation for a missing Delta expression in the visceral
mesoderm might be that a different factor acts as a ligand for Notch in the
visceral mesoderm and that the observed phenotype in Delta mutants is
due to secondary effects.
As the ectopic expression causes such a severe phenotype we also tested the
lethality of these embryos (Fig.
2). Most of the progeny of the cross between the bap-GAL4
driver line and UAS-N+Dl or UASNintra
develop and hatch but die as first larvae (78% or 70%), presumably owing to
the fact that they cannot ingest any food. Ectopic expression of
UAS-Dl alone also increased lethality compared with the UAS
and GAL4 lines alone (data not shown), but still 65% of the
larvae survive.
|
From these results, we conclude that Notch plays a role in the
determination of the founder cells and fcms in the visceral mesoderm.
Delta, which is expressed in the cells surrounding the visceral
mesoderm, might serve as the ligand in this process but it is also possible
that another factor takes over this role. Hence, not only is the fusion
mechanism between the founder cells and the fcms in the somatic and visceral
mesoderm conserved (San Martin et al.,
2001; Klapper et al.,
2002
), but so is the initial mechanism of determination of these
two cell types.
A screen for genes involved in visceral mesoderm development
To find out more about the mechanisms involved in the formation of the
visceral muscles, we decided to screen a collection of EMS mutagenised flies
(Hummel et al., 1999a;
Hummel et al., 1999b
) in order
to search for genes involved in the determination of the two visceral cell
types as well as in other aspects of visceral mesoderm differentiation.
Mutant embryos were stained and analysed with Fasciclin 3 (Fas3)
(Patel et al., 1987), which
marks the complete visceral mesoderm and allowed us to distinguish between the
two cell types. Founder cells show a strong Fas3 expression and are
characterised by a more columnar shape, while the more globular fcms show a
clearly weaker Fas3 expression (Klapper et
al., 2002
). Using this approach, we identified several mutants
with various defects in the development of the visceral musculature. These
novel mutants can be summed up in four subgroups
(Fig. 3).
|
Identification of jelly belly(jebweli) and the receptor for Jeb, milliways(miliAlk) the Drosophila Alk homologue
In the same screen, we found a fourth subgroup (G4) consisting of two
independent mutations, wellville (weli) and
milliways (mili), with the same, distinct phenotype
(Fig. 4). In these two mutants,
the continuous band of the visceral mesoderm in stage 11 is formed, but when
stained with Fas3, the more columnar shaped founder cells with the stronger
Fas3 expression are absent (Fig. 4B,C
compare with A). Thus, it appears that the founder cells of the
circular visceral muscles are not determined in either of these mutants. Using
the enhancer trap line rP298-lacZ, which shows a ß-galactosidase
pattern reflecting the expression of Duf/Kirre
(Nose et al., 1998;
Ruiz-Gómez et al.,
2000
), we could indeed show that in both mutants this founder cell
marker is not expressed in the visceral mesoderm
(Fig. 4E,F, compare with D). In
contrast to these observations, the determination of founder cells in the
somatic mesoderm is not affected (Fig.
4J-L), and the somatic muscle pattern shows only mild fusion
defects, which are especially obvious in the dorsal and ventral muscles
(Fig. 6A-C). At later stages no
visceral mesoderm is present in either mutant
(Fig. 4H,I, compare with
G).
|
|
mili displays the same distinct phenotype as jeb and we
reasoned that it is likely that both genes are involved in the same pathway.
As Jeb is a secreted protein the most promising candidate for mili
was Drosophila Alk, a member of the Alk/Ltk family of receptor
tyrosine kinases (RTKs), which is expressed in the nervous system and the
visceral mesoderm (Lorén et al.,
2001). Alk is considered to be a possible receptor for
jeb signalling (Lorén et
al., 2003
).
In order to further analyse whether mili is indeed an allele of
Alk, we tested a newly created deficiency in the region
(Df(2R)Alk21) in which Drosophila Alk has
been removed (Lorén et al.,
2003
). Indeed, mili is allelic to
Df(2R)Alk
21, and furthermore, embryos
transheterozygous for Df(2R)Alk
21 and mili
show the same phenotype as mili mutant embryos on Fas3 analysis (data
not shown). We then tested mili directly against the newly generated
Alk1 allele
(Lorén et al., 2003
)
and could indeed confirm that mili is a new Alk allele,
which we now named miliAlk. The analysis of
miliAlk mutants with the help of Alk antibodies
(Lorén et al., 2001
)
reveals that the mutant Alk protein is found in the cytoplasm instead of its
normal localisation at the cell membrane. Therefore we conclude that the
mutation is a phenotypic null allele. Furthermore, we could rescue the
specific loss of founder cells in the visceral mesoderm by ectopic expression
of UAS-Alk in the miliAlk mutant
background using bap-GAL4 as driver (data not shown).
Thus, the two newly identified mutants, both of which display the same, very distinct, phenotype of loss of founder cells in the visceral mesoderm, turn out to be novel jelly belly and Alk alleles.
The remaining cells of the visceral mesoderm in jebweli and miliAlk mutants differentiate to fusion-competent myoblasts
We have shown that the cells of the visceral mesoderm in
jebweli and miliAlk
mutants do not express the founder cell marker rP298-lacZ and exhibit
exclusively a globular shape upon Fas3 staining, which is characteristic for
fcms. This raised the question of whether the cells indeed are determined to
become fcms or remain undifferentiated. To clarify this question, we performed
in situ hybridisation with sns as probe
(Fig. 5). sns is
expressed in all fcms, both in the somatic and in the visceral mesoderm
(Bour et al., 2000;
San Martin et al., 2001
;
Klapper et al., 2002
). In the
wild type during stage 11, two bands of sns-expressing cells can be
observed in the mesoderm, which are connected in a ladder like pattern and
represent the fcms of the somatic and visceral mesoderm
(Fig. 5A). In
jebweli and miliAlk
mutants (Fig. 5B,C), only one
band is present whereas the other band is missing. As indicated by the
location of the connecting cells ventral of the present band, the dorsal band
consisting of the fcms of the visceral mesoderm is still present (arrowheads
in Fig. 5B,C). Thus, the
remaining cells in the visceral mesoderm differentiate as fcms and express
genes that are characteristic for this differentiated cell type.
|
Having found that sns is no longer expressed in the fcms of the
somatic mesoderm, we decided to look at the transcription factor lame
duck/myoblast incompetent/gleeful (lmd/minc/glee), which is
expressed in the somatic and visceral fcms and is responsible for their
determination (Duan et al.,
2001; Furlong et al.,
2001
; Ruiz-Gómez et
al., 2002
). The expression pattern of Lmd in the wild type is
similar to that of sns and the protein is present in two bands in
stage 11-12 (Fig. 5G). In both
jebweli and miliAlk
mutants, the Lmd expression pattern is present
(Fig. 5H,I) not only in the
fcms of the visceral mesoderm but also in the somatic ones, even though it
seems as if it is slightly weaker in the ventral part in the mutants than in
the wild type. These data suggest that in jebweli
and miliAlk mutants the initial determination of
the fcms in the somatic mesoderm takes place, but the subsequent
differentiation is blocked. Therefore, the Alk-RTK signalling pathway in the
somatic mesoderm seems to be essential for the differentiation of the fcms but
not for the initial determination.
The fusion-competent myoblasts of the visceral mesoderm become incorporated into the somatic mesoderm
Because most of the fcms of the somatic mesoderm are not expressing
sns in jebweli and
miliAlk mutants, we had a closer look at defects
in this tissue. ß-galactosidase antibody staining in mutants carrying the
founder cell marker rP298-lacZ show a regular pattern of somatic
founder cells compared with the wild type in the somatic mesoderm
(Fig. 4J-L). Only in some of
the mutant embryos could we detect local distortions because the defects in
the visceral mesoderm (data not shown). ß3tubulin antibody staining
(Leiss et al., 1988) shows
some mild fusion defects in the dorsal and ventral muscles in
jebweli and miliAlk
mutants indicated by unfused myoblasts in this region and long thin
projections of the muscles (Fig. 6B,C,
compare with A).
The development of the visceral mesodermal cells cannot be followed with
Fas3 staining because it disappears in the mutants after stage 11. Therefore,
we visualised the fate of the fcms using the visceral mesoderm marker
bap-lacZ, which normally is expressed exclusively in the visceral
mesoderm throughout embryonic development
(Azpiazu and Frasch, 1993;
Zaffran et al., 2001
)
(Fig. 6D). We observe that
jebweli and miliAlk
mutants carrying this marker show ß-galactosidase expression in the
somatic mesoderm from late stage 12 onwards
(Fig. 6E,F).
From previous studies, it is known that a lack of sns expression
in fcms in the somatic mesoderm results in strong defects in the somatic
musculature where the founder cells become blocked at the point of myoblast
fusion (Bour et al., 2000).
Because we are not able to detect such a strong phenotype in
miliAlk and jebweli
mutants, and together with the fact that bap-lacZ-expressing cells
are present in the somatic mesoderm, we conclude that the
sns-expressing cells from the visceral mesoderm now become
incorporated into the somatic mesoderm and replace at least a fraction of the
somatic fcms.
Alk protein is mislocalised in milliwaysAlk mutant embryos and co-localises with Jeb at the membranes of visceral founder cells
As jeb is a secreted protein we were interested whether the
localisation of Alk controls the specification for the more ventral cells of
the visceral mesoderm to become founder cells whereas the others develop into
fcms. Staining with anti-Alk antibodies
(Lorén et al., 2003)
show that in the wild type the protein is localised at the cell membranes in
the visceral mesoderm (Fig.
7A). Surprisingly, Alk can be found in the founder cells of the
circular visceral muscles and in the fusion-competent myoblasts
(Fig. 7A-C), which are not
obviously affected in miliAlk mutants
(Fig. 4C,F).
|
As Alk is localised at the membranes of all visceral cells and not only in
the founder cells, we reasoned that the localisation of the Jeb protein must
be responsible for the activation of the RTK pathway only in visceral founder
cells. Therefore we postulate a co-localisation of both proteins only at the
prospective founder cells. The double immunolabelling with Jeb
(Weiss et al., 2001) and Alk
(Loren et al., 2003
)
antibodies demonstrates that Jeb protein only co-localises at the membranes of
the visceral founder cells with the Alk protein
(Fig. 7J,K). Moreover, this
specific interaction cannot be found in miliAlk
mutants where, owing to the mislocalisation of the receptor protein, no Jeb
uptake takes place (Fig. 7L,M).
Therefore these mutants display an inactive RTK pathway.
Ectopic expression of UAS-jeb results in an increased number of founder cells in the visceral mesoderm
Previous work has shown that Jeb is secreted from the ventromedial cells of
the somatic mesoderm, which are close to the visceral mesoderm
(Weiss et al., 2001)
(Fig. 9). Because all cells of
the visceral mesoderm express the Alk RTK, it is theoretically possible that
all are able to respond to jeb signalling. The fact that only the
most ventral cells of the visceral mesoderm display an active RTK pathway as a
result of this interaction and later become the founders of the visceral
mesoderm could be explained by the fact that these cells are closest to the
cells that secrete the Jeb signal (Fig.
7K), which we suggest is the limiting factor. We therefore set out
to test whether increased levels of Jeb can change the fate of the more
dorsally located visceral fcms, which also express the receptor Alk, to become
founder cells. We again used the UAS-GAL4 system and expressed
UAS-jeb in the entire mesoderm with a twi-GAL4 driver. As
expected, nearly all cells of the visceral mesoderm are now converted to
founder cells (Fig. 8A,B). Even
though fcms are missing, the founders are able to form visceral muscles that
later on encircle the midgut. This ability to form muscles is one of the
characteristics of founder cells. From sns and mbc mutants,
it is known that even though no fusions take place, mini-muscles are formed in
the somatic mesoderm that display the right orientation and attachment sites
(Rushton et al., 1995
;
Bour et al., 2000
). This has
also been shown for the founder cells of the visceral mesoderm. In
sns mutants, apart from the first gut constriction the visceral
mesoderm develops normally (Bour et al.,
2000
). On closer inspection just the founder cells differentiate,
whereas the fcms remain undifferentiated
(Klapper et al., 2002
). Thus,
apart from the increased number of founder cells no defects are visible. The
same phenotype can be observed if UAS-jeb is ectopically expressed
only in the visceral mesoderm (data not shown).
|
|
In the wild type, the limited amount of the Jeb signal appears to restrict founder cell determination to the most ventral cells of the visceral mesoderm (Fig. 9). However, these findings prove that in principle all cells of the visceral mesoderm are able to respond to jeb signalling. Furthermore, we found no difference when the signal is produced from the somatic or the visceral mesoderm.
Ectopic expression of UAS-Alk gives a similar phenotype as in miliAlk and jebweli mutants
Anti-Alk stainings on embryos carrying the visceral mesoderm marker
bap-lacZ show that Alk is expressed in all cells of the visceral
mesoderm, some neuroectodermal cells and transiently in stage 10 to 11 in cell
clusters in the somatic mesoderm (Fig.
5D-F, Fig. 9). We
were interested in the consequences of ectopic expression of UAS-Alk
in the entire mesoderm. Surprisingly, the overexpression of UAS-Alk
by a twi-GAL4 driver produces a similar phenotype to that in
miliAlk or jebweli
mutant embryos. In early stage 11 only fcms are visible in Fas3 stainings
(Fig. 8C) and later on there is
no evidence of the presence of visceral mesoderm any more. In the somatic
mesoderm, defects can be seen by an anti-ß3tubulin antibody staining
(Fig. 8I). Several muscles are
small and display a spindle-like shape with long and thin projections,
indicating that only few myoblasts fuse to form the muscles. In comparison
with jebweli and
miliAlk mutants
(Fig. 6B,C), in the Alk
overexpressing embryos the muscle defects are stronger. Another surprising
finding was that in this overexpression situation the sns-expressing
cells of the somatic mesoderm are again missing
(Fig. 8F).
It remains an unanswered question why the overexpression of Alk in
the entire mesoderm results in a similar phenotype to that in
jebweli and miliAlk
mutants. One possible explanation for the visceral phenotype is that because
of the absence of sns-expressing cells in the somatic mesoderm,
jeb is not secreted anymore, which results in the absence of an
active RTK pathway in the visceral founder cells. Therefore, we carried out
anti-Jeb antibody stainings on these embryos. In stage 10 wild-type embryos,
jeb is expressed in two bands in the somatic mesoderm and disappears
in stage 12 from all mesodermal derivatives
(Weiss et al., 2001)
(Fig. 8L). In embryos
overexpressing Alk in the entire mesoderm, we could observe only one small
group of jeb-expressing cells per hemisegment
(Fig. 8M). The reduced amount
of the ligand Jeb thus phenocopies a jeb mutant situation where the
visceral founders are not determined.
A distinct difference between the founder cells and the fcms in the somatic
mesoderm is the expression of lmd/minc/glee in the fcms
(Duan et al., 2001;
Furlong et al., 2001
;
Ruiz-Gómez et al.,
2002
). We assume that in the wild type, only the fcms, which are
characterised by this expression, are able to respond to the Jeb/Alk
signalling pathway, which promotes the further differentiation of the somatic
fcms. These in turn continue to secrete Jeb, which is required for the
induction of the signalling pathway in the visceral mesoderm.
We assume that in the somatic mesoderm it is mainly the fcms that express Alk and suggest that the overexpression of Alk in the entire somatic mesoderm enables all cells of the mesoderm to take up the Jeb signal. Therefore, the signal necessary for the further differentiation of the fcms in the somatic mesoderm is downregulated through the increased Jeb uptake of the cells now ectopically expressing Alk. Another possibility to explain the visceral phenotype by overexpressing UAS-Alk in the whole mesoderm is that the overexpression of Alk itself leads to a strong downregulation of Jeb. As a consequence, the visceral founder cells are not specified, again owing to the lack of Jeb signal.
A further indication for the relevance of these changes in the somatic mesoderm for the visceral phenotype arises from the overexpression of Alk just in the visceral mesoderm with a bap-GAL4 driverline. This does not result in the phenotype described above. In this case, the founder cells in the visceral mesoderm are present and seem to be even increased in number (Fig. 8J-K). We assume that due to the Alk overexpression additional cells of the visceral mesoderm are now able to take up some of the limited amount of Jeb signal from the somatic mesoderm and thus become founder cells. In this case, Jeb expression in the somatic mesoderm is not affected.
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ACKNOWLEDGMENTS |
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Footnotes |
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