Research article 743

Myoblast determination in the somatic and visceral mesoderm

depends on Notch signalling as well as on milliways (miliAlk) as
receptor for Jeb signalling
Christiana Stute1, Kristina Schimmelpfeng2,*, Renate Renkawitz-Pohl1, Ruth H. Palmer3 and Anne Holz4,†
1Philipps-Universität Marburg, Fachbereich Biologie, Zoologie/Entwicklungsbiologie, Karl-von-Frisch-Strasse, 35039 Marburg,
Germany
2Institut für Neuro- und Verhaltensbiologie, Westfälische Wilhelms-Universität Münster, Badestrasse 9, 48149 Münster, Germany
3Umeå Center for Molecular Pathogenesis, Building 6L, Umeå University, S-90187, Sweden
4Institut für Allgemeine und Spezielle Zoologie, Allgemeine Zoologie und Entwicklungsbiologie, Justus-Liebig-Universität Gießen,
Stephanstraße 24, 35390 Gießen, Germany
*Present address: HHMI/Division of Cellular and Molecular Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA-92093-0683, USA
†Author for correspondence (e-mail: [email protected])

Development 131, 743-754

Published by The Company of Biologists 2004
doi:10.1242/dev.00972

Accepted 7 November 2003

Summary
The visceral muscles of the Drosophila midgut consist of fusion occurs resulting in a complete disruption of visceral
syncytia and arise by fusion of founder and fusion- myogenesis. Subsequent characterisation of the mutations
competent myoblasts, as described for the somatic muscles. revealed that they are novel alleles of jelly belly (jeb) and
A single-step fusion results in the formation of binucleate the Drosophila Alk homologue named milliways (miliAlk).
circular midgut muscles, whereas a multiple-step fusion We show that the process of founder cell determination in
process produces the longitudinal muscles. A prerequisite the visceral mesoderm depends on Jeb signalling via the
for muscle fusion is the establishment of myoblast diversity Milliways/Alk receptor.
in the mesoderm prior to the fusion process itself. We Moreover, we demonstrate that in the somatic mesoderm
provide evidence for a role of Notch signalling during determination of the opposite cell type, the fusion-
establishment of the different cell types in the visceral competent myoblasts, also depends on Jeb and Alk,
mesoderm, demonstrating that the basic mechanism revealing different roles for Jeb signalling in specifying
underlying the segregation of somatic muscle founder myoblast diversity. This novel mechanism uncovers a
cells is also conserved during visceral founder cell crosstalk between somatic and visceral mesoderm leading
determination. not only to the determination of different cell types but also
Searching for genes involved in the determination and maintains the separation of mesodermal tissues, the
differentiation of the different visceral cell types, we somatic and splanchnic mesoderm.
identified two independent mutations causing loss of
visceral midgut muscles. In both of these mutants visceral Key words: Drosophila, Myogenesis, Visceral and Somatic muscles,
muscle founder cells are missing and the visceral mesoderm Founder cells, Fusion-competent cells, Myoblast fusion, Notch,
consists of fusion-competent myoblasts only. Thus, no Delta, lethal of scute, jelly belly, Alk, RTK signalling

Introduction activated by Tin in the dorsal mesoderm. bap induction occurs

The early events of mesoderm formation and specification have in segmental clusters that migrate in anterioposterior direction
forming a continuous band of visceral progenitor cells, which
been studied extensively (reviewed by Anderson, 1998),
finally surrounds the midgut. Whereas bap is expressed
whereas the molecular mechanisms leading to different
transiently in the visceral precursor cells to determine the
mesodermal cell types are less understood. The primordia of
circular midgut muscles, the forkhead domain factor binou
visceral, somatic and cardiac tissues are established at defined (bin) is activated downstream of bap and is necessary for the
positions in each segment (Borkowski et al., 1995; Azpiazu et differentiation of all visceral derivatives. Hence, in bap and
al., 1996; Riechmann et al., 1997; Hosono et al., 2003). The bin mutant embryos, the visceral mesoderm is partially
subdivision of mesodermal cells during gastrulation is the basis transformed into somatic mesoderm (Azpiazu and Frasch,
for the constitution of the main types of musculature in 1993; Zaffran et al., 2001).
Drosophila: the somatic and visceral muscles. The formation Recently, Weiss et al. (Weiss et al., 2001) identified a novel
of visceral midgut muscles depends on two homeobox genes, signalling process that induces visceral cell identities. The jelly
tinman (tin) and bagpipe (bap) (Bodmer, 1993; Azpiazu and belly (jeb) gene encodes a secreted protein that is produced
Frasch, 1993). Expression of bap, which is required for the from somatic mesodermal cells and taken up by visceral cells.
determination of the midgut visceral mesodermal anlage, is In jeb mutants, no visceral muscles develop due to a failure
744 Development 131 (4) Research article

of differentiation in the existing Bap-expressing visceral cytoplasmic catalytic domain (Yarden and Ullrich, 1988).
precursors. The emerging picture shows, on the one hand, Ligand binding to the extracellular domain induces activation
visceral determination genes such as tin, bap and bin, which of the kinase on the cytoplasmic side, which initiates the
are responsible for subdividing the mesoderm into different intracellular signalling. The activated RTKs phosphorylate
tissues and, on the other hand, inductive signals like Jeb, which themselves and cytoplasmic substrates, leading to activation of
promote interactions between these tissues. a number of downstream signalling molecules, and ultimately
The differentiated visceral musculature of the Drosophila induce changes in gene expression and the phenotypic state of
midgut consists of an inner layer of circular muscles and the cell (Fantl et al., 1993; van der Geer et al., 1994). RTKs
an outer layer of longitudinal muscles (Campos-Ortega thus play important roles in cellular proliferation and
and Hartenstein, 1997), the latter persisting throughout differentiation. The role of RTKs during embryonic
metamorphosis (Klapper, 2000). The founder cells of the development and especially during the determination of
longitudinal muscles have a distinct primordium at the distinct cell types has been studied in detail in Drosophila
posteriormost part of the mesoderm (Tepass and Hartenstein, (reviewed by Rebay, 2002). During specification of muscle
1994; Georgias et al., 1997; Kusch and Reuter, 1999), whereas progenitor cells from equivalent cell clusters, the Ras/MAPK
the circular visceral founder cells and all fusion-competent pathway functions as an inductive cellular determination
myoblasts (fcms) for both visceral muscle types originate from signal. This pathway is activated by both epidermal and
the trunk mesoderm (Klapper et al., 2002). Recently, it has fibroblast growth factor receptors in the dorsal embryonic
been shown that both types of visceral muscles are syncytial mesoderm [(Gabay et al., 1997); DER (Buff et al., 1998) and
and arise by fusion of founders and fcms (Klapper et al., 2001; htl (Michelson et al., 1998)], while Notch antagonises this
San Martin et al., 2001; Klapper et al., 2002). These two activity by lateral inhibition (Carmena et al., 2002). Recently,
classes of myoblasts are closely associated in a band of visceral a novel RTK named DAlk (Drosophila Anaplastic Lymphoma
precursors, which express markers such as Fasciclin III (Patel Kinase; Alk – FlyBase) was described which is expressed
et al., 1987) and are characterised by binou expression (Zaffran specifically in the developing visceral mesoderm and CNS of
et al., 2001). The ventralmost row of these visceral myoblasts Drosophila (Lorén et al., 2001). Furthermore, activation of Alk
consists of dumbfounded/kin of irre (duf/kirre)-expressing mediated signalling is required for embryonic gut development
founder cells with a characteristic columnar shape. The more and more specifically for the activation of MAP kinase in the
dorsally located fcms are characterised as such by a more visceral mesoderm (Lorén et al., 2003). As Alk drives MAPK
globular morphology and by expression of sticks and stones activation in the visceral mesoderm it is an obvious candidate
(sns). During stage 12, binucleate circular muscles are built via to be involved in determination of distinct visceral cell types.
fusion of these founders and fcms. This fusion process is In a search for genes involved in the determination of
disturbed in myoblast city (mbc), duf/kirre and sns mutants, visceral cell types, we screened a collection of EMS-induced
which are also known to be defective in somatic muscle fusion, lethal mutations established previously (Hummel et al., 1999a;
indicating that the founder cell hypothesis applies both to Hummel et al., 1999b) and identified two independent
somatic and visceral myogenesis. Thus, several of the known mutations with a nearly identical phenotype. Both exhibit a loss
genetic components are common between somatic as well as of circular visceral founder cells at early stages of visceral
visceral myoblast fusion (Klapper et al., 2001; San Martin et development, whereas fusion-competent visceral myoblasts are
al., 2001; Klapper et al., 2002). specified correctly and express markers like Fas3 and Sns. This
Differentiation of the syncytial somatic muscles depends on results in a complete absence of visceral midgut muscles.
the determination of founder and fusion-competent myoblasts Complementation analysis revealed that one mutation belongs
(Bate, 1990; Dohrmann et al., 1990). These two classes of to the previously described jelly belly (jeb) gene, while the
myoblasts are specified by lateral inhibition by the neurogenic second mutation named milliways (miliAlk) represents a Alk
genes Notch and Delta from a group of equivalent somatic allele. We describe the crucial role of these genes in the process
mesodermal cells (Carmena et al., 2002). As a consequence, of visceral founder cell determination and analyse the role of
expression of the proneural gene lethal of scute (l’sc) is Notch and Delta during the distinction between visceral cell
restricted to muscle progenitor cells. These progenitors divide types. Moreover, we uncover Jeb signalling via the Alk RTK
asymmetrically and give rise to muscle founder cells (Carmena as a new determination step for the fusion-competent cells of
et al., 1995), which are characterised by differential expression the somatic mesoderm.
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
Materials and methods
precursor cells and in a second fusion step to formation of Flystocks
mature myotubes (Doberstein et al., 1997; Rau et al., 2001). To find out whether Notch and Delta mutants have defects in founder
This process can be disturbed at distinct levels (reviewed cell determination in the visceral mesoderm, we used N55e11 and DlB2
by Dworak and Sink, 2002; Taylor, 2002). Besides lateral mutants from the Bloomington Drosophila Stock Center. For analysis
inhibition and determination through distinct transcription of the involvement of Notch and Delta in the founder determination
process we used a dominant-negative form of Notch [UAS-dnNotch
factors cell-cell signalling is an important mechanism in (Rebay et al., 1993; Go et al., 1998)], UAS-Notch+Delta
myogenesis. (Bloomington Drosophila Stock Center, M. Muskavitch, unpublished)
Receptor tyrosine kinases (RTKs) are involved in and UAS-Notchintra flies (Lieber et al., 1993). UAS-Notchintra allows
intercellular communication in a wide range of processes. expression of a constitutive active form of the Notch receptor.
RTKs are composed of three domains: an extracellular ligand- The collection of EMS mutagenised flies was obtained from
binding domain, a single membrane-spanning domain and a Christian Klämbt (Hummel et al., 1999a; Hummel et al., 1999b). To
 Myoblast determination depends on N and Alk 745

screen for mutants with defects in the determination of founder cells myoblasts (fcms) of the somatic musculature (Carmena et al.,
in the visceral mesoderm, we stained 180 lines on the second and 270 2002). As many of the processes involved in the development
lines on the third chromosome, with anti-Fas 3 antibodies (Patel et al., of the somatic musculature also seem to affect the development
1987). We isolated a new jeb allele, jebweli and a new Alk allele, which of the visceral muscles, we investigated whether the
we named miliAlk. These two mutants were used for all experiments mechanism of determination of founder and fcms is also
described herein.
For complementation tests we used jebk05644 (Weiss et al., 2001)
conserved.
from the Bloomington Drosophila Stock Center, and Df(2R)Alk∆21 Notch and Delta play important roles in various
which is a deficiency covering 53C (Lorén et al., 2003). β- developmental processes and mutations in either gene lead to
galactosidase-expression in founder cells was achieved using the strong developmental defects during embryogenesis. Thus, it
enhancer trap line rP298-lacZ (Nose et al., 1998; Klapper et al., 2002) is difficult to analyse whether the visible defects in the visceral
and in the entire mesoderm using bap-lacZ (Azpiazu and Frasch, mesoderm are due to defects in the determination of the
1993; Zaffran et al., 2001). Overexpression studies were carried out founder cells or are a result of secondary effects. In Notch
using a bap-GAL4 driver line (Zaffran et al., 2001), which drives mutant embryos more founder cells appear to be present in the
expression in the circular visceral mesoderm from stage 10 onwards visceral mesoderm (Fig. 1A,B). The visceral fcms seem to be
or a twist-GAL4 driverline (SG24-GAL4), which drives expression in reduced compared with the wild-type expression of sticks and
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).
stones (sns) as a marker for these cells (Fig. 1D,E). This
All overexpression studies were carried out at 25°C. reduction is not as severe as in the somatic mesoderm but still
quite obvious. In Delta mutants, the number of founder cells
Immunohistochemical staining also seems to be increased in comparison with the wild type
Immunostaining was performed as described previously (Knirr et al., and the fcms are reduced in mutant embryos (Fig. 1C,F).
1999; Klapper et al., 2002). The mouse Fas3 antiserum (Patel et al., These observations cannot exclude the possibility that the
1987) was used for the visualisation of the visceral mesoderm cells observed phenotypes are induced by secondary effects from
(diluted 1:5, gift from C. Klämbt). A rabbit anti-β-galactosidase defects in other tissues, among others the lack of fcms in the
antibody (Biotrend, diluted 1:2500) was used to visualise muscle somatic mesoderm. We therefore decided to perform
founder cells in the enhancer trap line rP298-lacZ and a mouse anti- overexpression studies using the UAS-GAL4 system (Brand
β-galactosidase antibody (Promega, diluted 1:500) was used to
visualise bap-lacZ expression. For analysis of the somatic mesoderm,
and Perrimon, 1993). The GAL4 and UAS lines employed here
we used rabbit anti-β3tubulin antibodies at a dilution of 1:2500 (Leiss also carry rP298-lacZ (Nose et al., 1998), which serves to mark
et al., 1988). The rabbit anti-Alk (diluted 1:500) (Lorén et al., 2003), the founder cells. As a driver line we used bap-GAL4 (Zaffran
rabbit anti-Jeb (diluted 1:100) (Weiss et al., 2001), rabbit anti-Lmd et al., 2001) to drive expression in the entire trunk visceral
(diluted 1:1000) (Duan et al., 2001), mouse anti-Notch (diluted 1:10) mesoderm. Expression of UAS-Notch+Delta, which contains
(Fuß and Hoch, 2002) and mouse anti-Delta antiserum (diluted 1:50, the entire coding regions of both genes (M. Muskavitch,
Developmental Studies Hybridoma Bank) were used in combination unpublished) or UAS-Notchintra, which represents a
with the TSA signal amplification kit (NEN). For secondary constitutively active form of Notch (Lieber et al., 1993), in the
antibodies, we used Cy2- and Cy3-labelled antibodies made in goat visceral mesoderm, both result in a distinct phenotype. In
against rabbit and mouse from Dianova (diluted 1:40 and 1:100). The midgut preparations of these embryos the founder cells of the
embryos were embedded in Fluoromount G (Southern Biotechnology
Associates) and photos were taken under Nomarski optics with a Zeiss
circular visceral mesoderm are strongly reduced (Fig. 1K,L;
Axiophot microscope or a Leitz confocal microscope and processed data not shown for UAS-Notch+Delta) and later on, no
with Adobe Photoshop 6.0 (Adobe Systems). functional visceral mesoderm can be observed (data not
shown). By contrast, the founder cells of the longitudinal
In situ hybridisation visceral muscles, which have a different origin at the posterior
In order to visualise fcms in the mesoderm, whole-mount fluorescent tip of the embryo are still present (arrowheads in Fig. 1K,L).
DNA hybridisation was performed with random digoxigenin-labelled Interestingly, bap-GAL4-driven expression of the Notch ligand
sticks and stones cDNA probes according to Knirr et al. (Knirr et al., Delta does not result in fewer founder cells in the visceral
1999). The sns cDNA was a gift from S. Abmayr (Bour et al., 2000). mesoderm (data not shown).
For the combination with antibody stainings to visualise β- To exclude the possibility that the described defects are due
galactosidase expression we used anti-β-galactosidase antiserum
(Cappel) at a dilution of 1:3000.
to non-endogenous effects induced by the overexpression of
the examined genes in the wrong tissue, we analysed wild-type
Lethal phase analysis Notch expression and found that it is indeed expressed in the
To test the lethality of the progeny from UAS-GAL4 crosses we mated visceral mesoderm. Notch is localised at cell membranes in the
homozygous lines carrying the founder cell marker rP298-lacZ, entire visceral mesoderm during stage 11, with expression
collected eggs for 24 hours and allowed further development for becoming weaker in the fcms of the visceral mesoderm, which
another 48 hours at 25°C. Afterwards, we counted at least 1000 continue to express bap-lacZ after the determination process is
progeny of each cross. finished (Fig. 1G,H, arrowhead in G). This reduction of Notch
expression in the fcms after the establishment of the founder
Results and discussion cells is similar to its expression in the somatic mesoderm,
where Notch expression is also highest in the progenitor cell
Founder cell determination in the visceral after the determination process is completed (Carmena et al.,
mesoderm depends upon Notch 2002). Surprisingly, the analysis of Delta expression exhibits
The process of lateral inhibition involving Notch and its ligand that this Notch ligand is not expressed in the visceral mesoderm
Delta, which was first discovered in the nervous system, also during founder cell formation. Delta expression was found
plays a role in determining the founder and fusion-competent in adjacent, probably somatic cells (Fig. 1I,J) and might be
746 Development 131 (4) Research article

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 UAS-
Nintra 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.
To confirm these results, we
overexpressed a dominant-negative form of
Notch [UAS-dnN (Rebay et al., 1993; Go et
al., 1998)] specifically in the visceral
mesoderm with a bap-GAL4 driverline.
The embryos exhibit an obvious
duplication of most visceral founder cells
but still some fcms remain (Fig. 1M).
From these results, we conclude that
Fig. 1. Notch signalling is involved in founder cell determination in the visceral mesoderm. Notch plays a role in the determination of
The visceral mesoderm is visualised by Fas3 (red in A,B,C,M), β-galactosidase expression the founder cells and fcms in the visceral
from bap-lacZ (red in H,J) and founder cells are marked by rP298-lacZ expression (green in mesoderm. Delta, which is expressed in the
A,C,M brown in K,L). (A) Visceral mesoderm (vm) of a stage 11 wild-type embryo. (B) In cells surrounding the visceral mesoderm,
Fas3 staining of Notch55e11 embryos, it seems as if most cells of the vm are converted into might serve as the ligand in this process but
founder cells as indicated by the stronger Fas3 expression and the more rectangular shape. it is also possible that another factor takes
(C) In stage 11 DeltaB2 mutants, the number of founder cells also seems to be increased in over this role. Hence, not only is the fusion
the visceral mesoderm and fewer fcms are visible (compare C with A). The clusters of mechanism between the founder cells and
rP298-lacZ-positive cells ventral to the visceral mesoderm belong to the somatic mesoderm the fcms in the somatic and visceral
(sm). (D-F) sns in situ hybridisation comparing the wild type (D) where sns is expressed in
two bands with the sns expression in N55e11 (E) and DlB2 (F) embryos where it is reduced.
mesoderm conserved (San Martin et al.,
This reduction is more severe in the ventral band of the fcms of the somatic mesoderm but 2001; Klapper et al., 2002), but so is the
also visible in the dorsal band of the fcms of the visceral mesoderm. (G,H) Notch expression initial mechanism of determination of these
in the visceral mesoderm in stage 11 embryos. (G) Notch is expressed ubiquitously at the two cell types.
membrane of all cells of the visceral mesoderm. (H) Some cells seem to have a lower
expression level of Notch (arrowhead in G). These cells also express bap-lacZ, which at this A screen for genes involved in
stage is mainly restricted to the fcms (H). (I,J) In contrast to the N expression Delta is visceral mesoderm development
expressed in the cells adjacent to the bap-lacZ positive cell clusters of the visceral To find out more about the mechanisms
mesoderm. (K) rP298-lacZ expression in the gut of a stage 14 wild-type embryo. (L) In gut involved in the formation of the visceral
preparations of stage 14 embryos with ectopic expression of UAS-Nintra in the visceral muscles, we decided to screen a collection
mesoderm, the number of rP298-lacZ positive founder cells of the circular visceral
musculature is decreased. Founder cells of the longitudinal visceral muscles are not affected
of EMS mutagenised flies (Hummel et al.,
and migrate normally in anterior direction (arrow in L). (M) In stage 11 embryos 1999a; Hummel et al., 1999b) in order
overexpressing a dominant-negative form of Notch (UAS-dnN) with a bap-GAL4 driverline, to search for genes involved in the
the number of rP298-lacZ positive founder cells is increased compared with the wild type determination of the two visceral cell types
(A) and also some cells which are not marked by lacZ expression exhibit a stronger Fas3 as well as in other aspects of visceral
expression, which is characteristic for the founder cells in the visceral mesoderm. mesoderm differentiation.
Mutant embryos were stained and
needed there to participate in the visceral determination analysed with Fasciclin 3 (Fas3) (Patel et al., 1987), which
process, as indicated by the increased number of founder cells marks the complete visceral mesoderm and allowed us to
and reduced number of fcms in Delta mutants. Even though Dl distinguish between the two cell types. Founder cells show a
is expressed in the cells surrounding the visceral mesoderm, strong Fas3 expression and are characterised by a more
ectopic expression of UAS-Dl in these cells with a twi-GAL4 columnar shape, while the more globular fcms show a clearly
driver line does not result in an obvious phenotype (data not weaker Fas3 expression (Klapper et al., 2002). Using this
shown), which might be due to the fact that the amount of Delta approach, we identified several mutants with various defects in
in this tissue is not the limiting factor that restricts Notch the development of the visceral musculature. These novel
signalling. Another explanation for a missing Delta expression mutants can be summed up in four subgroups (Fig. 3).
 Myoblast determination depends on N and Alk 747

Fig. 2. Ectopic expression of Dl, Nintra and N+Dl in the visceral

mesoderm results in increased lethality. Lethality and survival rate of
the progeny of the UAS-GAL4 crosses (n≈1000). All crosses were Fig. 3. An EMS screening approach identifies novel mutations,
carried out with a bap-GAL4 line (asterisk indicates crossed to bap- which display severe defects during visceral mesoderm development.
GAL4) carrying rP298-lacZ as a founder cell marker. The development of the visceral mesoderm is visualised by Fas3
expression. (A-C) Wild-type development at stages 11 (A), 12 (B)
and stage 15 (C, arrowhead shows the midgut encircled by the
visceral mesoderm). (D-F) Examples for the different subgroups of
The first group (G1) shows no Fas3 staining in the visceral mutations are shown. (D) In embryos of the first subgroup (G1), no
mesoderm at any stage of embryonic development when visceral mesoderm can be detected at stage 11 (arrowhead). (E) In
compared with the wild type (Fig. 3A,D). In these G1 mutants, the second group (G2), embryos develop the initial patches of the
the initial subdivision of the mesoderm in precursors of the circular visceral muscles, but these patches subsequently fail to form
somatic and visceral mesoderm might be defective. The second a continuous band (arrowhead). (F) In the third group (G3), the
group (G2) shows Fas3 staining in the visceral mesoderm, but continuous band is formed but the cells of the visceral mesoderm do
not migrate dorsally and ventrally to encircle the entire midgut
the cells fail to form a continuous band (Fig. 3E) as is observed (arrowhead in F compare with arrowhead in C).
in wild type from stage 11 onwards (Fig. 3B). The third group
(G3) shows defects at even later stages. Here, the continuous
band of the visceral mesoderm forms, but the cells do not
migrate dorsally or ventrally (Fig. 3F) and thus do not encircle mesoderm is not affected (Fig. 4J-L), and the somatic muscle
the midgut as they do in wild-type embryos (arrowhead in Fig. pattern shows only mild fusion defects, which are especially
3C). It remains unclear whether this is due to a failure of obvious in the dorsal and ventral muscles (Fig. 6A-C). At later
myoblast fusion or to other reasons. The analysis of these stages no visceral mesoderm is present in either mutant (Fig.
mutants will be described elsewhere, while the fourth group 4H,I, compare with G).
(G4) is described here. Both mutations, weli and mili, are located on the second
chromosome. Complementation tests were subsequently
Identification of jelly belly (jebweli) and the receptor performed with mutants on the second chromosome, which are
for Jeb, milliways (miliAlk) the Drosophila Alk known to affect visceral mesoderm development. Surprisingly,
homologue this analysis revealed that weli is a new jelly belly (jebweli)
In the same screen, we found a fourth subgroup (G4) consisting allele. jeb encodes a secreted protein that is produced in the
of two independent mutations, wellville (weli) and milliways somatic mesoderm but is needed for proper visceral mesoderm
(mili), with the same, distinct phenotype (Fig. 4). In these two formation and has been proposed to be essential for the
mutants, the continuous band of the visceral mesoderm in stage migration and differentiation of the visceral mesoderm (Weiss
11 is formed, but when stained with Fas3, the more columnar et al., 2001). The phenotype of the specific loss of founder cells
shaped founder cells with the stronger Fas3 expression are of the circular visceral muscles has not been described.
absent (Fig. 4B,C compare with A). Thus, it appears that the mili displays the same distinct phenotype as jeb and we
founder cells of the circular visceral muscles are not reasoned that it is likely that both genes are involved in the
determined in either of these mutants. Using the enhancer trap same pathway. As Jeb is a secreted protein the most promising
line rP298-lacZ, which shows a β-galactosidase pattern candidate for mili was Drosophila Alk, a member of the
reflecting the expression of Duf/Kirre (Nose et al., 1998; Ruiz- Alk/Ltk family of receptor tyrosine kinases (RTKs), which is
Gómez et al., 2000), we could indeed show that in both mutants expressed in the nervous system and the visceral mesoderm
this founder cell marker is not expressed in the visceral (Lorén et al., 2001). Alk is considered to be a possible receptor
mesoderm (Fig. 4E,F, compare with D). In contrast to these for jeb signalling (Lorén et al., 2003).
observations, the determination of founder cells in the somatic In order to further analyse whether mili is indeed an allele
748 Development 131 (4) Research article

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
Fig. 4. In jebweli and miliAlk mutants the founder cells of the circular visceral
musculature are not determined. The development of the visceral mesoderm in wild- indicated by the location of the connecting cells
type (WT), jebweli and miliAlk mutants as visualised by Fas3 expression (red in A-I). ventral of the present band, the dorsal band
(A-F) Stage 11, (G-I) stage 15-16 and (J-L) stage 14. The nuclei of the founder cells consisting of the fcms of the visceral mesoderm
are marked by rP298-lacZ expression (green in D-F, brown in J-L). In jebweli and is still present (arrowheads in Fig. 5B,C). Thus,
miliAlk mutants at stage 11, the more columnar-shaped founder cells are missing. Only the remaining cells in the visceral mesoderm
cells that display the more globular shape of the fusion-competent myoblasts (fcm) differentiate as fcms and express genes that are
are present (B,C,E,F). Additionally, the founder cell marker rP298-lacZ is absent in characteristic for this differentiated cell type.
the visceral mesoderm in these mutants (E,F). The rP298-lacZ-positive cells, which
can be observed over the fcms of the visceral mesoderm in D-F belong to the somatic Jeb signalling and Alk expression are
mesoderm. At later stages, there are no signs of visceral musculature in either mutant also responsible for fusion-competent
(stage 16, arrowheads in H-I). (J-L) The founder cells of the somatic mesoderm are myoblast differentiation in the somatic
not affected in these mutants as visualised by rP298-lacZ expression (stage 14, K,L
mesoderm
compare with J).
The finding that in jebweli and miliAlk mutants in
addition to the lack of founder cells in the
of Alk, we tested a newly created deficiency in the region visceral mesoderm also the fcms of the somatic mesoderm do
(Df(2R)Alk∆21) in which Drosophila Alk has been removed not express fcm specific genes like sns was interesting as only
(Lorén et al., 2003). Indeed, mili is allelic to Df(2R)Alk∆21, mild defects in the somatic muscles are observed (Fig. 6B,C).
and furthermore, embryos transheterozygous for To explain this phenotype, we took a closer look at the Alk
Df(2R)Alk∆21 and mili show the same phenotype as mili expression in wild-type embryos. In addition to the expression
mutant embryos on Fas3 analysis (data not shown). We then of Alk in the cells of the visceral mesoderm, additional patches
tested mili directly against the newly generated Alk1 allele can be found in the neuroectoderm and the somatic mesoderm
(Lorén et al., 2003) and could indeed confirm that mili is a new during stages 10 and 11 (Fig. 5D-F). We conclude that these
Alk allele, which we now named miliAlk. The analysis of miliAlk patches of Alk expression in the somatic mesoderm are
mutants with the help of Alk antibodies (Lorén et al., 2001) essential for the development of the somatic fcms because in
reveals that the mutant Alk protein is found in the cytoplasm Alk mutant embryos, which are unable to activate the RTK
instead of its normal localisation at the cell membrane. pathway, these cells do not express fcms-specific genes (Fig.
Therefore we conclude that the mutation is a phenotypic null 5C). Furthermore, jeb signalling is also required for this
allele. Furthermore, we could rescue the specific loss of process, because the same phenotype can be observed in jeb
 Myoblast determination depends on N and Alk 749

mutants (Fig. 5B). Therefore the RTK signalling pathway Having found that sns is no longer expressed in the fcms
involving Jeb and Alk is not only needed for founder cell of the somatic mesoderm, we decided to look at the
specification of the visceral mesoderm but also for the transcription factor lame duck/myoblast incompetent/gleeful
differentiation of the fcms in the somatic mesoderm. (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
Fig. 5. The fusion-competent myoblasts of the somatic mesoderm do not differentiate in differentiation of the fcms but not for the
jebweli and miliAlk mutants. (A-C) sns in situ hybridisation of stage 11 embryos marks fcms initial determination.
in the visceral and somatic mesoderm. In wild-type embryos, sns is expressed in two bands
along the entire length of the embryo (A). These bands are connected in a ladder-like The fusion-competent myoblasts
pattern, where the ventral band represents the fcms of the somatic mesoderm (arrow in A), of the visceral mesoderm become
and the dorsal band consists of the fcms of the visceral mesoderm (arrowhead in A). In incorporated into the somatic
jebweli (B) and miliAlk (C) mutant embryos, the fcms of the visceral mesoderm and some mesoderm
cells that connect the two bands are present (arrowheads); however, the ventral band of fcms Because most of the fcms of the somatic
of the somatic mesoderm shows no sns expression. (D-F) Alk (green) and Fas3 expression
(red) in stage 11 wild-type embryos. In addition to expression in the visceral mesoderm, Alk
mesoderm are not expressing sns in jebweli
is also transiently expressed in some cells in the neuroectoderm and in patches in the and miliAlk mutants, we had a closer look
somatic mesoderm (arrows in D,F). (G-I) Lmd expression of stage 12 embryos. In the wild at defects in this tissue. β-galactosidase
type, Lmd is expressed in two bands in the fcms of the somatic and visceral mesoderm (G). antibody staining in mutants carrying the
In jebweli (H) and miliAlk (I) mutants, expression in both mesodermal cell types can be founder cell marker rP298-lacZ show a
observed. 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). β3tubulin 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
Fig. 6. The cells of the visceral mesoderm of jebweli and miliAlk mutants become mutants after stage 11. Therefore, we
incorporated in the somatic musculature. Ventrolateral (A,B,C) muscles of stage 16 embryos visualised the fate of the fcms using the
as visualised by β3tubulin expression (green in A-F). Cells of the visceral mesoderm are
marked by bap-lacZ expression (red in D-F). Compared with the wild type (A) the somatic
visceral mesoderm marker bap-lacZ,
muscles in both jebweli and miliAlk mutants are thinner and show long thin projections (B,C; which normally is expressed exclusively
arrow in B). Several unfused myoblasts are visible (arrow in C). (D-F) In wild-type in the visceral mesoderm throughout
embryos, no bap-lacZ expression is found in the somatic mesoderm during all stages of embryonic development (Azpiazu and
development (stage 14, D). By contrast, jebweli and miliAlk mutant embryos exhibit bap-lacZ Frasch, 1993; Zaffran et al., 2001) (Fig.
expression in the lateral muscles of the somatic mesoderm (stage 14, E,F). 6D). We observe that jebweli and miliAlk
750 Development 131 (4) Research article

mutants carrying this marker show β-galactosidase expression whereas the others develop into fcms. Staining with anti-Alk
in the somatic mesoderm from late stage 12 onwards (Fig. antibodies (Lorén et al., 2003) show that in the wild type the
6E,F). protein is localised at the cell membranes in the visceral
From previous studies, it is known that a lack of sns mesoderm (Fig. 7A). Surprisingly, Alk can be found in the
expression in fcms in the somatic mesoderm results in strong founder cells of the circular visceral muscles and in the fusion-
defects in the somatic musculature where the founder cells competent myoblasts (Fig. 7A-C), which are not obviously
become blocked at the point of myoblast fusion (Bour et al., affected in miliAlk mutants (Fig. 4C,F).
2000). Because we are not able to detect such a strong In jebweli mutants, the localisation of Alk is not affected.
phenotype in miliAlk and jebweli mutants, and together with the Identical to the wild type, it localises at the cell membranes
fact that bap-lacZ-expressing cells are present in the somatic and is also present in all cells of the visceral mesoderm, which
mesoderm, we conclude that the sns-expressing cells from the persist in these mutants (Fig. 7D-F). In miliAlk mutant embryos,
visceral mesoderm now become incorporated into the somatic however, the Alk protein is still detectable in all cells of the
mesoderm and replace at least a fraction of the somatic fcms. visceral mesoderm, but it is not correctly localised at the cell
membrane and is instead found in the cytoplasm (Fig. 7G-I).
Alk protein is mislocalised in milliwaysAlk mutant Because of this mislocalisation and the fact that the embryos
embryos and co-localises with Jeb at the transheterozygous for Df(2R)Alk∆21 and miliAlk display the
membranes of visceral founder cells same phenotype in the visceral mesoderm as the miliAlk mutant
As jeb is a secreted protein we were interested whether the embryos alone (data not shown), we conclude that the mutation
localisation of Alk controls the specification for the more is a phenotypic null allele even though at least the N-terminal
ventral cells of the visceral mesoderm to become founder cells part of the protein, against which the antibody is raised, is still
present. As the Alk receptor is not properly
localised, the founder cells cannot receive
the Jeb signal and thus the signal
transduction pathway leading to the
activation of duf/kirre in the visceral
founders is disturbed.
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 mis-
localisation 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
Fig. 7. Alk protein is mislocalised in miliAlk mutant embryos. Stage 11 embryos stained mesoderm
with Fas3 (B,E,H) to mark all cells of the visceral mesoderm or Alk antibodies (green in Previous work has shown that Jeb is
A,D,G). The stainings are merged in C,F,I. In wild-type embryos (A-C), Alk is expressed in secreted from the ventromedial cells of the
both the founder cells (f) and fusion-competent myoblasts (fcms) of the visceral mesoderm somatic mesoderm, which are close to the
and localises at the cell membrane. (A-C). In jeb mutant embryos (D-F), Alk is also
expressed at the membranes of all visceral cells. In miliAlk mutants, the majority of the
visceral mesoderm (Weiss et al., 2001)
protein does not localise at the cell membranes but instead can be found in the cytoplasm (Fig. 9). Because all cells of the visceral
(G-I). (J-M) In stage 11 wild-type embryos (J,K) Jeb (green in J-M) is taken up by the Alk- mesoderm express the Alk RTK, it is
expressing cells of the visceral mesoderm (red in K,M), whereas in miliAlk mutants (L,M) theoretically possible that all are able to
where the Alk protein is present but non-functional there is no co-localisation of the two respond to jeb signalling. The fact that only
proteins. the most ventral cells of the visceral
 Myoblast determination depends on N and Alk 751

mesoderm display an active RTK pathway as a result of this visceral muscles that later on encircle the midgut. This ability
interaction and later become the founders of the visceral to form muscles is one of the characteristics of founder cells.
mesoderm could be explained by the fact that these cells are From sns and mbc mutants, it is known that even though no
closest to the cells that secrete the Jeb signal (Fig. 7K), which fusions take place, mini-muscles are formed in the somatic
we suggest is the limiting factor. We therefore set out to test mesoderm that display the right orientation and attachment
whether increased levels of Jeb can change the fate of the more sites (Rushton et al., 1995; Bour et al., 2000). This has also
dorsally located visceral fcms, which also express the receptor been shown for the founder cells of the visceral mesoderm. In
Alk, to become founder cells. We again used the UAS-GAL4 sns mutants, apart from the first gut constriction the visceral
system and expressed UAS-jeb in the entire mesoderm with a mesoderm develops normally (Bour et al., 2000). On closer
twi-GAL4 driver. As expected, nearly all cells of the visceral inspection just the founder cells differentiate, whereas the fcms
mesoderm are now converted to founder cells (Fig. 8A,B). remain undifferentiated (Klapper et al., 2002). Thus, apart from
Even though fcms are missing, the founders are able to form 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).
sns in situ hybridisation confirms that
through the overexpression of UAS-jeb in
either the entire or just the visceral
mesoderm, the fate of the fcms is changed
so that they no longer express fcms-
specific gene products such as SNS. This
seems to be the reason why the band of
fcms of the visceral mesoderm (Fig. 8D)
is missing in these embryos (Fig. 8E). The
somatic mesoderm (Fig. 8G) shows no
defects as indicated by an anti-β3tubulin
staining (Fig. 8H). Overexpression of
UAS-jeb in a Alk mutant background
shows that the UAS-jeb overexpression
phenotype is suppressed in the Alk
mutants (data not shown). Therefore jeb
is dependent upon Alk as a receptor
to activate the downstream signalling
pathway.
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
Fig. 8. Ectopic expression of UAS-Alk and UAS-jeb in the mesoderm gives rise to different difference when the signal is produced
phenotypes. Wild-type embryos are shown in A,D,G and L. (B,E,H) UAS-jeb is ectopically from the somatic or the visceral
expressed with a twi-GAL4 driverline. (C,F,I,J,K,M) UAS-Alk is ectopically expressed with mesoderm.
a twi-GAL4 (C,F,I,M) or bap-GAL4 driver line (J,K). Founder cells (f) are marked by
rP298-lacZ expression (green in A,B,J,K). (D-F) Fusion-competent myoblasts (fcms) are Ectopic expression of UAS-Alk
visualised by sns in situ hybridisation. (B) Ectopic expression of UAS-jeb converts all cells
of the visceral mesoderm to founder cells. (E) sns expression is absent in these cells of the
gives a similar phenotype as in
visceral mesoderm. (H) The somatic mesoderm shows no defects, as indicated by β3tubulin miliAlk and jebweli mutants
antibody staining. (C) No visceral founder cells can be detected by Fas3 staining of stage Anti-Alk stainings on embryos carrying
11 embryos in which UAS-Alk is ectopically expressed in the entire mesoderm. (F) sns in the visceral mesoderm marker bap-lacZ
situ hybridisation indicates that through the overexpression of UAS-Alk the fcms of the show that Alk is expressed in all cells
somatic mesoderm are missing in stage 11 and only the band of the fcms of the visceral of the visceral mesoderm, some
mesoderm remains. (I) The dorsal and ventral somatic muscles in stage 16 show a fusion neuroectodermal cells and transiently in
defect phenotype with thin projections (arrow) and unfused myoblasts (arrowhead) as stage 10 to 11 in cell clusters in the
indicated by β3tubulin staining. When UAS-Alk is expressed only in the visceral mesoderm
with a bap-GAL4 driverline, which also carries rP298-lacZ as founder cell marker, the
somatic mesoderm (Fig. 5D-F, Fig. 9). We
founder cells of the visceral mesoderm are still present and seem to be doubled in number were interested in the consequences of
(J,K). In contrast to a single row in the wild type (A), a second row of founder cells is ectopic expression of UAS-Alk in the
present in these stage 11 embryos. The bands of both halves of the embryo are shown (J,K). entire mesoderm. Surprisingly, the
In the wild type, jeb is expressed in a continuous band in the mesoderm (L), whereas upon overexpression of UAS-Alk by a twi-GAL4
overexpression of UAS-Alk in the entire mesoderm the signal is reduced (M). driver produces a similar phenotype to
752 Development 131 (4) Research article

S 10 stronger. Another surprising finding was that in this

overexpression situation the sns-expressing cells of the somatic
mesoderm are again missing (Fig. 8F).
bap bap vm 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
sm
pathway in the visceral founder cells. Therefore, we carried out
anti-Jeb antibody stainings on these embryos. In stage 10 wild-
ecto 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
S 11 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
fcm’s phenocopies a jeb mutant situation where the visceral founders
sns sns vm
are not determined.
duf/kirre
f duf/kirre 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
fb et al., 2002). We assume that in the wild type, only the fcms,
fcm sns sns sns which are characterised by this expression, are able to respond
sm
f f f
to the Jeb/Alk signalling pathway, which promotes the further
differentiation of the somatic fcms. These in turn continue to
ecto 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
jeb/milliAlk fcms that express Alk and suggest that the overexpression of
jeb/Jeb milliAlk
Alk in the entire somatic mesoderm enables all cells of the
Fig. 9. The mode of action of Mili/Alk-mediated Jeb signalling mesoderm to take up the Jeb signal. Therefore, the signal
during the determination and differentiation within the visceral and necessary for the further differentiation of the fcms in the
somatic mesoderm. At stage 10 (S 10) ventromedial somatic somatic mesoderm is downregulated through the increased Jeb
precursor cells start to express jelly belly (jeb, red cells) and secrete uptake of the cells now ectopically expressing Alk. Another
Jeb protein (small red dots) (Weiss et al., 2001). Some of these cells possibility to explain the visceral phenotype by overexpressing
(red with green crescents) also express miliAlk (green) and all visceral UAS-Alk in the whole mesoderm is that the overexpression of
precursor cells express miliAlk and bagpipe (bap) at this time. We Alk itself leads to a strong downregulation of Jeb. As a
conclude that Jeb signalling, especially the RTK pathway activation consequence, the visceral founder cells are not specified, again
in visceral founder cells, depends on the level of Jeb protein reaching
these cells. This activation leads in stage 11 (S 11) in the visceral
owing to the lack of Jeb signal.
mesoderm (vm) to the expression of dumbfounded/kin of irre A further indication for the relevance of these changes in the
(duf/kirre, blue) in the visceral founder cells (f), whereas the fusion- somatic mesoderm for the visceral phenotype arises from the
competent myoblasts (fcms) without an active pathway are overexpression of Alk just in the visceral mesoderm with a bap-
characterised by sticks and stones (sns) expression. We further GAL4 driverline. This does not result in the phenotype
suggest that initial jeb expression in the somatic mesoderm (sm) is described above. In this case, the founder cells in the visceral
maintained mainly in lame duck/myoblast incompetent/gleeful mesoderm are present and seem to be even increased in number
(lmd/minc/glee)-expressing fcms responding on RTK pathway (Fig. 8J-K). We assume that due to the Alk overexpression
activation with differentiation into sns-expressing fcms. Nothing is additional cells of the visceral mesoderm are now able to take
known so far about the role of Jeb and Mili/Alk signalling in the up some of the limited amount of Jeb signal from the somatic
ectoderm (ecto) and fat body (fb).
mesoderm and thus become founder cells. In this case, Jeb
expression in the somatic mesoderm is not affected.

that in miliAlk or jebweli mutant embryos. In early stage 11 only We are grateful to Christian Klämbt for sharing fly stocks and
fcms are visible in Fas3 stainings (Fig. 8C) and later on there results prior to publication. We thank Peter Seum for excellent
is no evidence of the presence of visceral mesoderm any more. technical assistance. We are indebted to Simone Lier, Henning Mootz
and Christian Klämbt for critical reading of the manuscript and
In the somatic mesoderm, defects can be seen by an anti-
helpful comments. We thank the Bloomington Stock Center for fly
β3tubulin antibody staining (Fig. 8I). Several muscles are small stocks, Christian Klämbt for Fas3 antibody, Manfred Frasch for bap-
and display a spindle-like shape with long and thin projections, GAL4 and bap-lacZ, Joe Weiss for UAS-jeb flies and Jeb-antibody,
indicating that only few myoblasts fuse to form the muscles. Thomas Klein for UAS-Nintra flies, Dieter Maier for UAS-dnNotch
In comparison with jebweli and miliAlk mutants (Fig. 6B,C), in flies, Hanh Nguyen for Lmd-antibodies, Alan Michelson for SG24-
the Alk overexpressing embryos the muscle defects are GAL4 flies, Akinao Nose for rP298-lacZ, and Susan Abmayr for the
 Myoblast determination depends on N and Alk 753

generous gift of sns cDNA. This work was supported by the Deutsche subdivision of trunk visceral mesoderm parasegments in Drosophila is
Forschungsgemeinschaft to R.R.-P. (Re 628/10-3 and 10-4) as well as required for gut and trachea development. Development 130, 439-449.
the Kerckhoff-Stiftung and the Fonds der Chemischen Industrie to Hummel, T., Schimmelpfeng, K. and Klämbt, C. (1999a). Commissure
R.R.-P., and by a Landesgraduiertenstipendium nach dem Hessischen formation in the embryonic CNS of Drosophila. Dev. Biol. 209, 381-398.
Gesetz zur Förderung von Nachwuchswissenschaftlern to C.S. Hummel, T., Schimmelpfeng, K. and Klämbt, C. (1999b). Commissure
formation in the embryonic CNS of Drosophila. Development 126, 771-
779.
Klapper, R. (2000). The longitudinal musculature of Drosophila melanogaster
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