Laboratoire de Génétique et Physiologie du Développement, UMR 6545 CNRS-Université, IBDM CNRS-INSERM-Université de la Méditerranée, Campus de Luminy, 13288 Marseille Cedex 09, France
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Abstract |
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The gene encoding the subunit of the
Drosophila Go protein is expressed early in embryogenesis in the precursor cells of the heart tube, of the
visceral muscles, and of the nervous system. This
early expression coincides with the onset of the mesenchymal-epithelial transition to which are subjected
the cardial cells and the precursor cells of the visceral
musculature. This gene constitutes an appropriate marker to follow this transition. In addition, a detailed analysis of its expression suggests that the cardioblasts originate from two subpopulations of cells in
each parasegment of the dorsal mesoderm that might
depend on the wingless and hedgehog signaling pathways for both their determination and specification.
In the nervous system, the expression of Go
shortly precedes the beginning of axonogenesis.
Mutants produced in the Go gene harbor abnormalities in the three tissues in which the gene is expressed. In particular, the heart does not form properly and interruptions in the heart epithelium are
repeatedly observed, henceforth the brokenheart (bkh)
name. Furthermore, in the bkh mutant embryos, the
epithelial polarity of cardial cells was not acquired
(or maintained) in various places of the cardiac tube.
We predict that bkh might be involved in vesicular
traffic of membrane proteins that is responsible for
the acquisition of polarity.
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Introduction |
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IN Drosophila, the larval heart (or dorsal vessel) is a
simple linear tube composed of inner myoendothelial
cells (cardial cells) and outer unpolarized and nonmyogenic cells (pericardial cells) (Rizki, 1978; Rugendorff
et al., 1994
; Bodmer, 1995
; Bodmer et al., 1997
). It extends
from the T2 thoracic segment to the A7 abdominal segment. The dorsal vessel is surrounded by a network of extracellular matrix components (Rugendorff et al., 1994
), among which some are localized in specialized areas of its
surface (Zaffran et al., 1995
). The heart behaves as a pump
to insure a continuous flow of hemolymph in the organism.
The heart precursor cells arise from a group of ventral
blastoderm cells in the presumptive mesoderm anlagen
which invaginates at gastrulation (Bate, 1993). Later, the
mesoderm spreads and forms a monolayer of cells in close
apposition to the ectoderm in the direction of the dorsal
region of the embryo. Meanwhile, the cells divide three to
four times during the process. A proper dorsal-lateral migration is controlled by the activity of heartless, a gene encoding a fibroblast growth factor receptor (Beiman et al.,
1996
; Gisselbrecht et al., 1996
). Once the mesodermal cells have reached their ultimate location, they require inductive signals from the overlying ectoderm to accomplish
both their determination and differentiation. The progenitors of the cardial cells derive from a particular region in
the mesoderm that receives simultaneously the ectodermal signals of the Decapentaplegic (Dpp) and the Wingless (Wg) secreted proteins and that constitutes the cardiogenic region (Staehling-Hampton et al., 1994
; Frasch,
1995
; Lawrence et al., 1995
; Wu et al., 1995
; Bodmer et al.,
1997
).
In stage 11 embryos, the precursor cells of the cardiac
lineage are metamerically organized in groups of mesenchymal cells that do not, however, fully mirror the
structure of the mature heart (see for example Dunin-Borkowski et al., 1995; Zaffran et al., 1995
; Riechmann et
al., 1997
). From stage 11 onwards and at the beginning of
germ-band retraction, the heart cell progenitors acquire
their specification by expressing a combinatorial set of
marker genes specific for each cell subpopulation.
We are interested in the genetic control of cardiogenesis
in Drosophila which is viewed as a simple model to study
organogenesis. The cardial founder cells become specified
in two subpopulations from a unique mesodermal functional domain and further differentiate into cells exerting
markedly different functions that are the myoendothelial
cells (the cardial cells) and the pericardial cells. Along this
determination/differentiation process, the cardial cells
acquire a polarity that is the result of a typical mesenchyme-epithelium transition. To better understand these
transformations at the molecular level, we have focused
our attention on genes that are specifically expressed in
distinct subpopulations of heart cells such as the gene encoding the subunit of the Go protein. Indeed, an expression of this gene early in embryogenesis in the precursors of the cardial cells has been described (Zaffran et al.,
1995
) in addition to its expression in the embryonic nervous system (Guillèn et al., 1991
; Wolfgang et al., 1991
).
The G proteins, which are heterotrimeric GTPases, behave as signal transduction proteins and they are widespread proteins that have been conserved from yeast to
humans. Their three subunits ,
, and
form a complex
that relay external signals inside the cell via a receptor
(serpentine receptor) composed of seven transmembrane
segments, activable by a variety of ligands, including hormones or neuropeptides. In the inactive complex, GDP is
bound to the
subunit and activation of the receptor results in an exchange between GDP and GTP and in the
dissociation of the heterotrimer in a monomer (
subunit)
and a dimer (
and
subunits). Both entities can be partners in the regulation of the activity of various enzymes
and of ionic channels that in turn control the level of intracellular second messengers (see for review Clapham and
Neer, 1993
; Neer, 1995
).
In Drosophila, genes coding for proteins that belong to
the three major families of subunits (
s,
i, and
o) have
been cloned and characterized. Two Gs
proteins differing
by four amino acids are produced by alternative splicing of
a unique gene (Quan and Forte, 1990
). The unique gene
for Gi
produces two transcripts which encode the same
protein (Provost et al., 1988
). Finally, two Go
proteins
have been deduced from cDNA sequence analyses and are
issued from a unique gene transcribed in three different transcripts (de Sousa et al., 1989
; Schmidt et al., 1989
;
Thambi et al., 1989
; Yoon et al., 1989
). Two other
subunits have been identified as homologues of vertebrate
Gf
(Quan et al., 1993
) and Gq
-3 (Talluri et al., 1995
), respectively. All these proteins are expressed throughout
embryogenesis in various tissues in a dynamic pattern
(Wolfgang et al., 1991
) and, consequently, may play major
roles in development although no precise developmental function has been ascribed to any of them. In contrast, it is known that the activity of concertina which encodes the
subunit of a G12 or G13 protein (Wilkie and Yokoyama,
1994
) is necessary for the coordination of cell shape
changes in the ventral furrow at gastrulation (Parks and
Wieschaus, 1991
). In addition, it is a likely candidate to act
downstream of a putative secreted protein, Fog, whose receptor is as yet unidentified (Costa et al., 1994
).
Among the different families of G proteins, the Go protein is perhaps the less well characterized in terms of the
nature of its associated signal transduction pathway. On
the plasma membrane, Go proteins may regulate Ca2+ and
K+ channels (Van Dongen et al., 1988; Warnick et al.,
1988
; Kleuss et al., 1991
; Llédo et al., 1992
), distinct types
of phospholipase C (Moriarty et al., 1990
), and the mitogen-activated protein kinase cascade (Van Biesen et al.,
1996
). In addition to interacting with heptahelical receptors such as opiate,
2-adrenergic, D2 dopaminergic, and
somatostatin receptors, the Go protein has been described
recently as a regulator of vesicular traffic (Lagriffoul et al.,
1996
; Gasman et al., 1998
).
In Drosophila, the amount of Go protein is affected in
memory mutants (Guillèn et al., 1990) and is expressed in
the nervous system during development at the onset of axonal growth (Guillèn et al., 1991
; Wolfgang et al., 1991
).
We show here that the function of Go is required for the
formation of the heart, the visceral musculature, and the
nervous system. Based on the phenotype observed in mutants for the gene encoding the
subunit of the Go protein, we have chosen brokenheart (bkh) as its name.
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Materials and Methods |
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Generation and Analysis of Mutations
The Df(2R)47A deficiency in the bkh gene region was issued from a cross
between AQ65 (a P-element (P[w+, lacZ]) enhancer trap line in 47A) homozygous males which were -irradiated (4,000 rad) and (w; Sp/Cyo) females. Males and females from the F1 generation which were Cyo, but not
Sp, were crossed together to obtain the strain. Among the 5,000 chromosomes which were screened, only one line affected the bkh mRNA expression: Df(2R)47A.
Three P-lines P[w, lacZ] (Török et al., 1993) l(2)k06915, l(2)k11003,
and l(2)k07810, inserted on the second chromosome, had been located in
the 47A region and were homozygous lethal. PCR analysis of the P-lines'
genomic DNA was used for mapping these insertions. The primers were
the 31-bp terminal sequence of the P-element (5'-CGACGGGACCACCTTATGTTATTTCATCATG-3') and bkh specific primers: (bkh6: 5'-CAGCCCATTGTGGTTGGG-3'; bkh3: 5'-AAGTCCTCAGCAGTGAAGCCGCTCTCG-3'). P07810 was localized 1.5 kb upstream of the class
II cDNA ATG and the 1.5-kb PCR fragment obtained was sequenced by
Genome Express. P06915 and P11003 were localized 800 bp upstream of
the class I cDNA ATG. All the mutant stocks were balanced with Cyo
(wg, lacZ) to identify the homozygous mutant embryos.
A genomic region in the 5' end of the bkh gene was identified with the aid of the DS01583 P1 phage obtained from the Drosophila Genome Project. The 5' end of the DS01583 P1 phage was located 4.5 kb downstream of the class II cDNA ATG. Amplifications of genomic sequences from this region were performed with the pair of specific primers bkh6 and bkh16 (bkh16 5'-CTGAACTTCGAGCGTCGTC-3'). Sequences were made by Genome Express and analyzed with Gene Finder to identify transcription units.
The bkh007 mutation was generated by P-element mobilization in the
P07810 line and a cross with flies bearing (2-3) as a source of transposase
and of w; Sp/Cyo;
(2-3),Sb/TM6B genotype.
P-Element-mediated Transformation
Germline transformation was performed using standard procedures as described by Rubin and Spradling (1982). The bkh cDNA (class II) was subcloned in the pUAST vector (Brand and Perrimon, 1993
) and injected at a
concentration of 300 ng/µl with the
(2-3) helper plasmid (100 ng/µl)
(Robertson et al., 1988
). Several independent transformants were obtained and mapped, and lines of interest were made homozygous with the
help of a double balancer stock w; Sp/CyO; MKRS/TM2.
Rescue with the GAL4 System
To rescue the Df(2R)47A phenotype at the embryonic stage, we used the
yeast GAL4-directed transcription system (Brand and Perrimon, 1993).
Females homozygous for a UAS-8.31bkh class II cDNA on the X chromosome (UAS-bkh) and bearing the Df(2R)47A deficiency were crossed to
homozygous males bearing the homozygous 24B-GAL4 driver inserted on
the third chromosome and the Df(2R)47A deficiency on the second chromosome. Homozygous animals were sorted out with the aid of a labeled
CyO balancer and scored by observation of their phenotypes.
Immunohistochemistry and In Situ Hybridization
Digoxigenin (DIG)1-labeled antisense or sense RNA probes were generated from DNA with T3 or T7 RNA polymerase (Promega) and DIG-UTP (Boehringer). They were used for whole mount in situ hybridization of fixed staged embryos prepared as described in Zaffran et al. (1997).
The DIG-labeled RNA probes were detected with the aid of a preadsorbed anti-DIG antibody coupled to alkaline phosphatase (Boehringer)
and NBT/BCIP as substrate. The embryos were mounted in Geltol medium (Immunotech) for further observation. A specific probe for class I
bkh cDNA was obtained by PCR amplification of genomic DNA with the
oligonucleotides bkh4 and bkh7. A probe for class II cDNA was prepared
by amplification of the cDNA inserted in pBlueScript with the T7 primer
and the bkh6 oligonucleotide (bkh4: 5'-GCCCATGGTGCGTATTGCCTCGACGC-3'; bkh7: 5'-CCGCTAGCGGTCAAATCGGGTATCCT-3'; bkh6, see above).
For immunohistochemistry experiments, embryos were fixed and
stained with antibodies according to the protocol described by Ashburner
(1989).
-Galactosidase in embryos was detected by using a mouse anti-
-galactosidase (Promega) antibody diluted 1,000-fold. The following primary antibodies were used: anti-BKH (1:100 dilution; Guillèn et al., 1991
);
anti-DMEF2 (1:2,000 dilution; Bour et al., 1995
); anti-EC11 (1:2 dilution;
Zaffran et al., 1995
); anti-Fas II, anti-Fas III, anti-Eve and anti-En (1:10,
1:2, 1:500, and 1:10 dilutions, respectively; Developmental Studies Hybridoma Bank); anti-
-spectrin (1:500 dilution; Lee et al., 1993
); anti-Nrt (1:
500 dilution; Piovant and Lena, 1988
) and anti-Tin (1:800 dilution; Azpiazu and Frasch, 1993
). Affinity-purified secondary antibodies (Jackson
ImmunoResearch Laboratories) were either coupled to alkaline phosphatase or Biotin and used at a 1:1,000 dilution or conjugated to TRITC
or FITC and used at a 1:100 dilution. The stained embryos were mounted
in Geltol medium (Immunotech) for further observation under an Axiophot Zeiss microscope or, when fluorescent, in Permafluor (Immunotech)
for observation under an Axiophot or a LSM 410 Zeiss confocal microscope.
In double labeling experiments, the conditions were the same as above. In situ hybridizations were performed before staining with antibodies. After dehydration of the embryos labeled with antisense RNA probes, they were rehydrated in PBS containing 0.3% Triton X-100 for at least 1 h and saturated in the same buffer containing 10% FCS before incubation with the primary antibodies.
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Results |
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The Dynamic Expression of bkh during Embryogenesis Suggests a Developmental Function
The organization of the gene brokenheart (bkh) encoding
the subunit of Go, as described by Yoon et al. (1989)
, is
schematized in Fig. 1. Two classes of cDNAs (class I and
class II) have been characterized (de Sousa et al., 1989
;
Schmidt et al., 1989
; Thambi et al., 1989
; Yoon et al.,
1989
). They arise from an alternative splicing in a unique
gene and differ only in the ATG-containing first exon.
Three developmentally regulated transcripts (3.4 kb of
maternal origin, 4.2 and 6 kb) were revealed (Wolfgang et al., 1991
) and by using specific probes, we have shown that the
class I cDNA corresponded to the 6-kb mRNA appearing
after 12 h of development whereas the class II cDNA corresponded to the 4.2-kb mRNA more abundant in earlier
stages (data not shown).
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Both cDNAs encoded proteins of the same size (354 amino acids) that diverged only by 7 amino acids among their 21 NH2-terminal residues and for which it is not known whether they exert different functions.
By using the class II cDNA as probe, a strong expression of bkh was observed in preblastoderm embryos due
to the presence of the 3.4-kb maternal transcript (not
shown). In early stage 11 embryos, the zygotic transcript
could be detected for the first time in clusters of cells
within 11 segments (Fig. 2, a and b). The cells appear to be
cardial and visceral muscle progenitor cells since, based on
bkh staining patterns in later stage 12 embryos, the cells
became integrated into the conspicuous monocellular layer of cardial and visceral mesoderm cells on each side
of the embryo (Fig. 2, c-e). This assumption was further
supported by bkh expression in embryonic tissues which
were unambiguously constituted of such cells (Fig. 2 e)
and, also, in tinman (tin) loss of function mutants. In tin
mutants, neither heart nor visceral muscles are formed
(Azpiazu and Frasch, 1993; Bodmer, 1993
) and, correlatively, bkh expression was completely abolished in the territories from which the precursor cells for these two tissues
originated (Fig. 2 g). From the middle of stage 11 onwards,
neuroblasts of the central nervous system (CNS) became
labeled (Fig. 2 c) and bkh expression persisted in the neurons of the CNS in later stages of embryogenesis (Fig. 2,
d-f). In a similar way, all the neurons of the peripheral
nervous system (PNS) expressed the bkh mRNA from
stage 12 onwards, slightly before the onset of axonogenesis (Fig. 2, d and f). Probes for either cDNA gave identical
spatial patterns of expression although class I transcripts
were quantitatively less abundant and were expressed
later than the class II transcripts (not shown).
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Antibodies directed against a COOH-terminal peptide
whose sequence was conserved in the subunit of all Go
proteins showed that the pattern of expression of the protein was superimposable to that of the mRNA during embryogenesis (Fig. 2, i and j). However, probably due to the
presence of the protein of maternal origin, these antibodies were poorly efficient in detecting a significant signal in
the cardial cells as early as did mRNA probes.
bkh Is an Early Marker of the Mesenchymal-Epithelial Transition Undergone by the Cardioblasts
In stage 11 embryos, the zygotic bkh mRNA was detected
at first in the precursors of the cardioblasts in a repeated
pattern of 11 clusters in the dorsal mesoderm (Fig. 3, a and
b). These clusters, constituted initially of two to four cells,
were located in the anterior compartment of each mesodermal parasegment in a position anterior to the domain
of En ectodermal stripes (Fig. 4) and closely neighboring
the Wg-expressing cells in parasegments 2-12 of the overlying ectoderm. These cardioblast precursors belonged to
the functional domain of Ladybird (Lbd) and of Evenskipped (Eve) expressing cells in the mesoderm which are
the precursors of a fraction of the pericardial cells and of a
dorsal muscle (see for example, Lawrence et al., 1995; Azpiazu et al., 1996
; Jagla et al., 1997
). Within the cluster, the
expression of the bkh mRNA was initially very intense in
only two of the cells and then it became detectable in the
four cells with an almost uniform intensity. Due to their
position relative to the parasegment boundary, we call
these cells A (for anterior) cardioblasts. Soon after the onset of bkh expression in the first cluster, a second cluster of
stained cells appeared posterior to the first one and not in continuity with it (Fig. 3, c and d). This group of cells
called P (for posterior) cardioblasts was located in the posterior domain of the next parasegment and it never contained more than two bkh-expressing cells. Their position
coincided with that of an area posterior to En stripes in the
ectoderm (Fig. 4).
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During germ-band retraction (late stage 11 and stage
12), the cardioblast progenitors that are mesenchymal in
nature at that stage began to establish contacts between
themselves, to extend filopodes (Rugendorff et al., 1994;
Dunin-Borkowski et al., 1995
), and to reorganize their
shape to form a continuous layer on each side of the dorsal
opening (Fig. 3, e-j). The origin of this process was the result of the encounter of the A and P groups of bkh-expressing cells in each parasegmental domain (Fig. 3, g
and h). Later in development and due to the shortening of
the segment during germ-band retraction, the segmentally
repeated six cell clusters spread along the anterior-posterior axis to finally join as a monolayer of polarized epithelial cells (Fig. 3, g-j). Still further in the process, the two
rows of cardial cells, together with the pericardial cells that
are attached to their basal membrane, fused at the dorsal
midline to form the heart tube. Our observations led to the
conclusion that a mature heart at the end of embryogenesis was constituted of 52 cardial cells per hemiembryo partitioned in six cells in every segment from T3 to A7 and in
only four cells in the T2 segment. The results described
above suggest the existence in each segment of two distinct populations of cells composed of four and two cardial
cells that derived from mesodermal cells and that were located in domains corresponding, respectively, to the anterior and posterior compartment of each parasegment (Fig.
4). None of the cardioblasts present in parasegments 2 and 3 and only a part of those found in PS4 participated in the
mature dorsal vessel, but it was, however, difficult to follow their fate since the expression of bkh vanished rapidly
from these cells during development.
Similarly, by using the EC11 antibody specific for the
pericardial cells (Zaffran et al., 1995), we counted 36 such
cells distributed in 4 cells per segment from T2 to A7. Eve-expressing cells from the labial segment and the segment
T1 did not contribute to the pool of pericardial cells and
they probably gave rise to other structures that we were
unable to recognize.
The cardial cells, which are also muscular cells, were determined as epithelial (Rugendorff et al., 1994; Tepass,
1997
), based on the expression of several markers for
polarity (see below). For example, antibodies directed
against
-spectrin specifically labeled the basal-lateral
membrane of the epithelium (Lee et al., 1993
) (see Fig. 8
a) and the staining by anti-Neurotactin antibodies (Nrt)
(Piovant and Lena, 1988
) was restricted to the lateral and apical domains of the cell surface (see Fig. 8 e). In contrast, phosphotyrosyl proteins (Woods et al., 1997
) and
Armadillo (Müller and Wieschaus, 1996
) were weakly expressed in the heart cell membranes and had no polarized
localization (not shown), in good agreement with the lack
of authentic Zonulae adherens in Drosophila secondary epithelia (Tepass and Knust, 1993
; Tepass and Hartenstein, 1994
). Finally, the basal membrane of the epithelium
was visualized with the mAb EC11 (Zaffran et al., 1995
).
This antibody recognized an antigen probably secreted by
the pericardial cells which was detected on the basal membrane of the cardial cells and around the pericardial cells
(see Fig. 8 a).
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In the visceral mesoderm that also undergoes a mesenchymal-epithelial transition before its differentiation into
the visceral musculature, bkh was expressed in a repeated
pattern of clusters of cells in the same 11 parasegments as
the precursors of the cardial cells and anterior to them
(data not shown; Azpiazu et al., 1996; Riechmann et al.,
1997
). These precursor cells in parasegments 4-12 were ultimately transformed in a polarized epithelial ribbon of
one cell in width (Bate, 1993
), whose polarity was assessed by the localization of Fasciclin III (Patel et al., 1987
) to
cell-cell contacts (see Fig. 8 i).
bkh Is Required for the Formation of the Cardial and of the Visceral Mesoderm Epithelia
The embryonic expression of bkh suggests a function of the protein Go in the formation of the cardial and visceral mesoderm epithelia as well as in axonal growth. A deficiency in the 47A region in which is localized the bkh gene was obtained by using as a starting tool the homozygous viable AQ65 line whose lac Z reporter gene was specifically expressed in the entire nervous system. Among the 5,000 chromosomes mutagenized by x-ray irradiation and screened for the loss of the phenotypic white marker, a lethal deficiency Df(2R)47A was obtained. It was shown by cytology that it had lost half of each of the A and B bands in 47 (not shown). Quantitative analyses of Western blots revealed that in animals heterozygous for that deficiency, half the amount of the BKH protein was produced as compared with its production in the initial AQ65 line (not shown). Moreover, no mRNA could be detected in homozygous mutant embryos (Fig. 2 h).
Three P-insertion lines, issued from the systematic
screen of lethal P-insertions on the second chromosome
(Török et al., 1993), have been located within the cytological region of the bkh gene. In the P07810 insertion, the lac
Z reporter gene expression was superimposable to that of
the bkh mRNA (Fig. 2 k), whereas, in the P06915 and
P11003 insertions, the
-galactosidase activity was restricted to the visceral mesoderm (Fig. 2 l). The different insertions were mapped by PCR in the 5' region of the
class II and class I cDNAs, respectively (see Fig. 1 and Materials and Methods). The lethality in these lines was not
consecutive to the insertion of the transposon but was
rather due to a secondary mutation since the homozygous
lethal lines were perfectly viable in trans of Df(2R)47A
and of other deficiencies uncovering the same region
[Df(2R)E3363, Df(2R)X1, Df(2R)Stan2, Df(2R)12].
In contrast, in the P07810 line, mobilization of the P-element has resulted in the obtention of several viable revertants and of one lethal bkh007 white revertant in trans of
Df(2R)47A. Homozygous bkh007 embryos had the same
heart and visceral muscle phenotype as that displayed by
the homozygous Df(2R)47A-deficient animals (see below).
To analyze the molecular lesions resulting from the imprecise excision of the P07810 transposon, the 5' region of the gene in which P07810 lies has been further characterized. A P1 phage (DS01583) covering the whole length of the bkh gene contained 4.5 kb of genomic sequence upstream of the class II cDNA ATG. This fragment was cloned, fully sequenced, and analyzed for a more detailed definition of the class II transcription unit. Three canonical potential TATA boxes were located within a 510-bp sequence downstream of the P07810 insertion site. The analysis of the bkh007 allele showed that a segment of 839 bp was deleted that contained the three potential TATA boxes together with 311 bp of the 5' end of the transcription unit. As a consequence, no class II mRNA was produced in the homozygous bkh007 embryos.
Homozygous Df(2R)47A embryos (recognized by the
lack of a labeled CyO balancer) harbored defects in the
three tissues in which bkh was expressed with a total penetrance meaning that all homozygous Df(2R)47A embryos
had defects in the heart, the visceral mesoderm, and the
nervous system. When probed with anti-EC11 antibodies (Zaffran et al., 1995), the mutant embryos showed interruptions in their dorsal vessel (Fig. 5, c and d) that were
also visible when DMef 2 expression was investigated in
all the muscle cells including the cardial cells (Fig. 5, f-h)
(Bour et al., 1995
). The cardial cells were unambiguously
present and they had migrated properly in dorsal position,
but, in some places, they were no longer arranged as a continuous layer but rather as unorganized clusters of cells
(Fig. 5, f-h). Fas III expression revealed the same types of
defects in the visceral mesoderm (Fig. 5 j). Similar alterations were detected in bkh007 mutants with the same total
penetrance (not shown).
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In the nervous system, longitudinal axons were often
missing (Fig. 6 b) and important modifications were observed in the guidance and axonal growth of motoneurons
(not shown). It should be recalled here that the lola gene
was also deleted in the Df(2R)47A deficiency and that
mutations in this gene result in missing longitudinal axons
in the CNS (Fig. 6 c; Seeger et al., 1993; Giniger et al., 1994
). Since the Df(2R)47A phenotype was slightly stronger (in terms of penetrance of the phenotype) than that
provoked by a loss of function of lola, this suggests a function per se for Go in axonal growth or guidance. This was
confirmed by the analysis of the bkh007 mutation which
fully complemented mutations in the lola gene but continued to display a neuronal phenotype. In homozygous
bkh007 mutants, axons of the motoneurons were clearly
misrouted (not shown). However, in contrast to the
Df(2R)47A mutation, the longitudinal axons in the CNS
were missing with a lower frequency but were often
pinched (Fig. 6 d).
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The heart and visceral mesoderm phenotypes could be
rescued at least partially by expressing the class II cDNA
under the control of 24B-GAL4 (Brand and Perrimon,
1993; Zaffran et al., 1997
) that drives the expression of
UAS-cDNAs in the myogenic lineage including the cardioblasts and the visceral mesoderm. In >65% (n = 250 observed embryos) of homozygous Df(2R)47A embryos, a
normal development of these tissues had been restored
(Fig. 7 a). In contrast, the phenotype in the nervous system
was not abolished (Fig. 7 b) as expected, first because the
deficiency uncovers the lola gene, and second because of
the restricted specificity of expression of the 24B-GAL4
line. The bkh007 mutation that affects only the bkh gene
could also be rescued by a bkh transgene (>80% of homozygous bkh007 embryos were rescued over a total of 147 embryos examined).
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bkh Is Required for the Acquisition of Epithelial Polarity of the Cardial and Visceral Mesoderm Cells
The loss of bkh function produced heart and visceral mesoderm phenotypes that suggest a participation of the Go
protein in epithelia formation. During the mesenchymal-epithelial transition, subsets of membrane and cytoskeletal
proteins localize to distinct regions of the cell surface to
create the apical and basal-lateral membrane domains
(Drubin and Nelson, 1996) that confer to the cells their epithelial polarity. The bkh mutant phenotype was more accurately described with the aid of markers localized specifically to these different domains. We focused our analysis
on regions of the mutant heart in which the cells formed a
continuous uninterrupted layer but which displayed abnormal marker expression.
As shown in Fig. 8, b and d, the localization of two polarity markers was affected in several places in the heart
epithelium of bkh mutant embryos. -Spectrin, specific for
the basal-lateral membrane of epithelial cells, was now expressed on the entire surface of the mutant cells, however
with a somewhat lower intensity than in wild-type cells. In
addition, these mutant cells remained round, had no signs
of shape remodeling, and failed to express the EC11 antigen in their basal membrane. The pericardial cells which
are normally attached to the basal membrane of the cardioblasts were absent from these same regions and were
rather often associated in clusters in the domains of high
EC11 antigen expression (not shown). Similarly, Nrt
which was localized to the apical and lateral membranes of
wild-type cardial cells (Fig. 8 e) was, in bkh embryos, scattered in several locations on the entire surface of those cells which were round and in which the epithelial array
was disorganized (Fig. 8, f and h). Finally, Fas III expression also revealed phenotypes in the visceral mesoderm
characterized in some regions by a uniform and lower Fas
III staining on the surface of still round cells with a partially destroyed epithelial structure (Fig. 8, j and l).
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Discussion |
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The brokenheart Gene Function Is Required for the Morphogenesis of the Heart and Visceral Musculature
In a deficiency, Df(2R)47A, which completely deleted the
brokenheart gene encoding the subunit of the heterotrimeric Go protein, the morphogenesis of the heart and of
the visceral mesoderm was impaired. The defects associated to the nervous system have not been studied in detail,
essentially because this deficiency also uncovered the longitudinal absent gene (lola) whose requirement for axonogenesis of longitudinal fascicles is known (Seeger et al.,
1993
; Giniger et al., 1994
). However, several arguments
strongly suggest that the phenotypes associated to the
heart morphogenesis and to the visceral musculature were
a consequence of a loss of the function of bkh. First, in deficient animals, alterations were restricted to the tissues
that expressed bkh and they appeared in synchrony with
the temporal expression of bkh. Second, P-element mobilization in enhancer trap lines located within the bkh gene and imprecise excision of the P-transposon led to the isolation of mutants with phenotypes identical to those observed in homozygous Df(2R)47A embryos. Finally, we
were able to rescue the heart and visceral mesoderm embryonic phenotypes in homozygous bkh mutants by expressing the cDNA encoding bkh in transgenic flies. The
failure to totally rescue the phenotype probably resulted
from the fact that 24B-GAL4 did not drive the expression
of the UAS-bkh cDNA in mesodermal cells early enough
to be totally efficient (Michelson, 1994
).
Although the heart was perturbed in the totality of the
mutant embryos (100% penetrance), defects were observed only in some of the cells that constituted the dorsal
vessel. This result does not support well the contention
that bkh is an essential partner in the acquisition of polarity for the epithelial cardial cells since, if this were the
case, all the cells would be equally affected. The maternal
protein which is abundant and stable until mid-embryogenesis could have partially compensated the loss of zygotic transcript. Production of germline clones lacking bkh
activity could help to elucidate this point. Moreover, another heterotrimeric Gi protein is expressed in the cardial
cells (Wolfgang et al., 1991) and functional redundancy
might have been at work.
The bkh Gene Function Is Required for the Acquisition of Cell Polarity in the Morphogenesis of the Heart and of the Visceral Musculature
The myoendothelial heart tube is considered as a secondary epithelium that forms by a mesenchymal-epithelial
transition (Rugendorff et al., 1994; Tepass and Hartenstein, 1994
; Zaffran et al., 1995
; Tepass, 1997
). The mesenchymal cardial precursor cells, after their migration from
the ventral site of gastrulation in the direction of their final
dorsal position, reorganize their plasma membrane to acquire their cellular polarity and establish cell junctions to
build up the cardial epithelium. A signal must be received by the mesenchymal cells to create the first asymmetry on
their surface (for review see Drubin and Nelson, 1996
). Interactions with localized extracellular matrix components
are believed to be largely responsible for this first event
(Drubin and Nelson, 1996
; Tepass, 1997
) and Drosophila
mutants, for example, in Laminin A (Yarnitzky and Volk,
1995
; Tepass, 1997
) or in an integrin subunit (Stark et al.,
1997
), present severe disruptions in their dorsal vessel.
The initiating signal is probably emitted in response to inductive interactions between the mesenchymal cells and
the overlying dorsal ectoderm. The newly created asymmetry could then trigger a reorganization of the cytoskeleton. Finally, the different membrane domains (apical and
basal-lateral) will be established by the acquisition of different combinatorials of membrane proteins that have
been specifically routed towards them; this sorting-out step relies on an absolute specificity of the vesicular traffic (for review see Rodriguez-Boulan and Powell, 1992
). In
the absence of bkh function, the mesenchymal cardial cells
did not remodel their shape and, in the case of the most
extreme phenotype, they failed to form a continuous row
of cells. In addition, whenever such a layer was eventually
formed, in several places some but not all cells neither acquired nor maintained a proper polarity.
Based on the timetable of bkh expression, its participation in the first step creating the asymmetry of the cardial cells is not very likely. It might rather be required for the subsequent steps that concern cell shape change and polarization by addressing membrane proteins to their respective domains.
It has been suggested that heterotrimeric G proteins
could contribute to the vesicular protein traffic by regulating early steps in the secretory pathway (for review see
Nuoffer and Balch, 1994). This hypothesis stems from the
observation that AlF4
, an activator of heterotrimeric but
not of monomeric G proteins, inhibits ER to Golgi and intra-Golgi transport as well as vesicle budding from the
trans-Golgi network. In particular, Go proteins have been
implicated in granule exocytosis from chromaffin cells (Vitale et al., 1993
; Gasman et al., 1998
), insulin secretion (Lang et al., 1995
), and transcytosis (Bomsel and Mostov,
1992
). It has been shown that the secretion of the protease
Nexin-1 by glioma cells was under the control of Go1 (Lagriffoul et al., 1996
). The Go
1 protein was detected on the
membrane of small intracellular vesicles and the secretion
of Nexin-1 was stimulated by Go
1 overexpression and by
activators of Go proteins such as mastoparan. It was further suggested that the GTPase activity of the Go
1 protein could be stimulated in the absence of a classical serpentine receptor (Lagriffoul et al., 1996
).
Thus, we are tempted to predict that the Drosophila Go protein has a function in a particular type of vesicular traffic responsible for the acquisition or maintenance of cell polarity in the cardial and visceral mesoderm cells. Preliminary observations on the subcellular localization of Bkh in embryonic cells were consistent with this prediction. In the totality of the cells examined, Bkh was located to the cytoplasm rather than associated to the cell membrane and the staining pattern revealed a typical granular appearance (data not shown).
Since the exportation and the localization to the plasma membrane of the protein markers we have used were all equally affected in bkh mutants, we conclude that bkh is involved in a general aspect of vesicular traffic rather than in the specific process of the sorting out of membrane proteins.
bkh might also be required for the reorganization of the
cytoskeleton. The protein G encoded by the concertina
gene participates in cell shape changes taking place at gastrulation (Parks and Wieschaus, 1991), probably via a
modulation of the invaginating blastoderm cell cytoskeleton resulting from the activation of RhoA (Hall, 1998
).
The early role of the Go protein in the formation of the
heart epithelium does not exclude a function in later
events leading to the formation of the heart or to the acquisition of its function. It has been shown recently that
knocking-out the Go gene in the mouse resulted in heart
dysfunction. Go-deficient mice had lost the muscarinic inhibition of isoproterenol-stimulated cardiac L-type Ca2+
currents (Valenzuela et al., 1997
; Jiang et al., 1998
). It will be interesting to investigate whether the Drosophila Go
protein could also be involved in such a process.
The Lineage of the Cardial Cells
Before the determination of cardiac precursors in the mesoderm, the overlying ectoderm is subdivided in segmentally repeated units partitioned into an anterior compartment (A-compartment) and a posterior compartment
(P-compartment). Analysis of the expression of genes involved in the specification of mesodermal derivatives and
other observations lend support to the idea that, after gastrulation, the mesoderm also becomes subdivided into segmentally repeated units, each of which consists of two separate domains (Dunin-Borkowski et al., 1995; Azpiazu et al.,
1996
; Riechmann et al., 1997
). The domains that are located below the ectodermal P-compartments are subject
to influences from the striped regulators eve and hh and have been termed "P-domains" or "eve-domains." By contrast, the development of the metameric domains that are
located below the A-compartments depends largely on the
striped regulators wg and slp (sloppy-paired) and these domains have been termed "A-domains" or "slp-domains."
In that scheme, three basic groups of genes are at work
to pattern the mesoderm either along the dorso-ventral
axis (dpp and tinman) to specify the dorsal mesoderm, or
along the anterior-posterior axis (wg and slp) to subdivide
it into segmental units, or at defined positions to control
tissue specification. For example, recent evidence suggests
that wg, whose expression is restricted to striped domains
in each of the A-compartments and which is required for a
variety of inductive signaling events during embryonic development, is directly involved in heart formation, in that it is necessary for further subdividing of the dorsal mesoderm and for specifying cardial cell fates. Elimination of
the wg function shortly after gastrulation, at a time when
tin becomes restricted to the dorsal mesoderm, results in
the selective loss of heart progenitor cells with little effect
on segmental patterning of the cuticle or other mesodermal derivatives (Baylies et al., 1995; Lawrence et al., 1995
;
Wu et al., 1995
; Azpiazu et al., 1996
; Park et al., 1996
).
From these and other observations, a picture has emerged
in which specification of precardiac and dorsal somatic muscle precursors requires intersections of the dorsal domains of dpp expression with the transverse stripes of the
dorsal expression of wg (Bodmer and Frasch, 1998
).
However, the results presented herein are merely consistent with some precursors of the cardial cells originating
from the P-domains and subjected to the influence of hh
signaling rather than to that of wg. This hypothesis is
somewhat difficult to verify because wg expression after
gastrulation requires hh and vice versa (Klingensmith and
Nusse, 1994), but, later in embryogenesis, the two signals
become independent. Therefore, we have investigated the
expression of Tin in temperature-sensitive hhts2 mutant
embryos submitted to a temperature shift ~5 h after egg laying (stage 10) (Fig. 9). In these embryos, the wild-type
expression of Tin in four cardial and four pericardial cells
was expanded to the two cardial cells located in the
P-domains of each segment. We predict that these two tin-expressing cells are the same as the two bkh-expressing
cells in this same domain.
|
The observation that the cardial progenitor cells can be
divided into two cell subpopulations is consistent with the
situation in the mature heart tube in which two genetically
distinct populations of cardial cells have been described
(Bodmer et al., 1997). For example, tin (see for example
Bodmer, 1993
; Jagla et al., 1997
) as well as
3-tubulin
(Leiss et al., 1988
) and several P-lacZ reporter genes from
enhancer trap lines (Hartenstein et al., 1992
) are expressed in only four cell pairs per segment among the six pairs present. In the same line, a D-mef2 enhancer element directed lacZ transcription in four cardial cell pairs
per segment consistent with a direct regulation by tin
which is expressed in these same cells (Gajewski et al.,
1997
). Interestingly, these two subpopulations of cardial
cells, respectively, reside below the anterior and the posterior ectodermal parasegmental domains (Fig. 4).
These different observations could mean that the two
P-cardioblasts were specified by the inductive instruction
of hh rather than by that of wg. However, it is not known
whether the hh pathway provides a direct late cardiogenic
signal or exerts its effect via suppressing the wg function in
the posterior domain. This hypothesis is unlikely in that, at
that stage, reducing the function of hh in the epidermis
does not lead to any visible effect on the wg signaling pathway (Heemskerk and Di Nardo, 1994). Indeed, wg expression in the dorsal epidermis was not expanded in a hh mutant (not shown). We predict then that hh might behave as
a repressive signal for tin expression in the two P-cardioblasts. Cell heterogeneity in terms of gene expression
could then be achieved along the anterior-posterior axis
by an efficient cooperativity of wg and hh signals in the
specification of the cardial cells.
![]() |
Footnotes |
---|
Address correspondence to M. Sémériva, Laboratoire de Génétique et Physiologie du Développement, UMR 6545 CNRS-Université, IBDM CNRS-INSERM-Université de la Méditerranée, Campus de Luminy, Case 907, 13288 Marseille Cedex 09, France. Tel.: 4-91-26-96-11. Fax: 4-91-82-06-82. E-mail: semeriva{at}lgpd.univ-mrs.fr
Received for publication 29 July 1998 and in revised form 14 April 1999.
Our thanks are due to Drs. Goodman, Frasch, Giniger, and Kiss for their
generous gifts of strains used in this work, to the Bloomington Stocks
Center that sent us stock flies and the BDGP that sent us the DS01583 P1
phage. We thank Drs. Kiehart, Piovant, Frasch, and N'Guyen for their
gifts of some of the antibodies mentioned herein. Anti-Fas II, -Fas III,
-En, and -Eve antibodies were provided by the Developmental Studies
Hybridoma Bank in Baltimore. We acknowledge S. Long and F. Graziani
for their technical assistance in the food preparation for the maintenance
of the stock flies. We are deeply indebted to Dr. D. Gratecos for her help
in the preparation and drafting of the manuscript.
S. Zaffran's present address is Brookdale Center for Developmental and
Molecular Biology, Mount Sinai School of Medicine, Box 1126, 1 Gustave
L. Levy Place, New York, NY 10029. A. Guillèn's present address is Departamento de Bioquimica y Biologia Molecular I, Facultad de Biologia,
Universidad Complutense, 28040 Madrid, Spain. V. Homburger's present
address is Centre CNRS-INSERM de Pharmacologie et Endocrinologie,
141, Rue de la Cardonille, F-34094, Montpellier Cedex 5, France.
This work was supported by the Centre National de la Recherche Scientifique and by grants from Association Française contre les Myopathies (AFM), Association pour la Recherche contre le Cancer (ARC) and Ligue contre le Cancer (LCC).
![]() |
Abbreviations used in this paper |
---|
CNS, central nervous system; DIG, digoxigenin; PNS, peripheral nervous system.
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