Department of Molecular Genetics, The Weizmann Institute of Science, 76100 Rehovot, Israel
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Abstract |
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In the Drosophila embryo, the correct association of muscles with their specific tendon cells is achieved through reciprocal interactions between these two distinct cell types. Tendon cell differentiation is initiated by activation of the EGF-receptor signaling pathway within these cells by Vein, a neuregulin-like factor secreted by the approaching myotube. Here, we describe the cloning and the molecular and genetic analyses of kakapo, a Drosophila gene, expressed in the tendons, that is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. A major defect in the kakapo mutant tendon cells is the failure of Vein to be localized at the muscle-tendon junctional site; instead, Vein is dispersed and its levels are reduced. This may lead to aberrant differentiation of tendon cells and consequently to the kakapo mutant deranged somatic muscle phenotype.
Key words: muscles; Drosophila; tendons; Egfr; neuregulin; cytoskeleton ![]() |
Introduction |
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DIFFERENTIATION of the somatic musculature of the Drosophila embryo is a complex, multistep process, resulting in a segmentally reiterated pattern of muscles that govern larval locomotion via muscle connections to discrete attachment sites in the epidermis. After initial periods of independent differentiation, the mesodermally derived muscle cells and the epidermal attachment cells use a complex signaling mechanism, through which connections between the two cell types and final differentiation are achieved.
During the second half of Drosophila embryogenesis,
each of the specific somatic myotubes extends its leading
edge towards a specific location, at which a group of epidermal muscle attachment (EMA)1 cells is located. At the
end of this extension process, each myotube forms a physical contact with a specific EMA cell, which is then induced
to develop into a mature tendon cell (Bate, 1993; Becker
et al., 1997
).
The larval tendon cells develop in the embryo in two sequential steps: initially, a subset of ectodermally derived
competent EMA cells is defined along the A-P and D-V
axes. In a second step, the portion of these competent cells
that are bound to muscles differentiate into mature tendon
cells (Becker et al., 1997). The expression of the regulatory
protein Stripe, a transcription factor of the early growth
response (EGR) family, determines the fate of the EMA
competent cells at the first phase of tendon cell development (Lee et al., 1995
; Frommer et al., 1996
). Stripe expression leads to the expression of an array of EMA-specific genes that contribute to the correct guidance of the
myotubes (Becker et al., 1997
; Vorbrüggen et al., 1997).
The second phase of tendon cell differentiation depends
on inductive interactions between the myotube and the
EMA cell. These interactions lead to terminal differentiation of the EMA competent cells into tendon cells, in
which high protein levels of Stripe, Groovin (Volk and VijayRaghavan, 1994), and Alien (Goubeaud et al., 1996
)
are maintained, and the transcription of the genes delilah
(Armand et al., 1994
) and
1 tubulin (Buttgereit et al., 1991
) is induced.
The inductive signal responsible for triggering the muscle-dependent differentiation of the tendon cells is provided by Vein, a secreted protein that is homologous to
vertebrate neuregulins (Schnepp et al., 1996). Vein is necessary and sufficient to induce the expression of tendon-specific genes, including stripe, groovin, delilah, and
1 tubulin (Yarnitzky et al., 1997
). Vein activity is mediated
through its activation of the Drosophila EGF receptor homologue, DER, expressed on the EMA cells (Yarnitzky
et al., 1997
; Schnepp et al., 1998
). Thus, Vein acts as a secreted differentiation factor that mediates the muscle-dependent differentiation of the EMA cells into tendon
cells.
Although vein mRNA is produced in the muscle cells,
Vein protein is highly concentrated in the intercellular
space between the muscles and the tendon cells, where intense adherens type junctions are formed (Yarnitzky et al.,
1997). This junctional space contains electron-dense material, which presumably represents protein aggregates
of various extracellular matrix components (Tepass and
Hartenstein, 1994
). Since the primary sequence of Vein includes a signal peptide but no transmembrane domain, it is
assumed that Vein protein is secreted from the myotube
and accumulates at the muscle-tendon junctional space.
The molecular mechanism that is responsible for Vein localization at this site is yet to be elucidated. The Vein
ligand is a relatively weak activator of the EGF receptor
pathway (Schnepp et al., 1998
; Yarnitzky et al., 1998
);
therefore, a mechanism regulating Vein accumulation at
the site of activity may be essential for a proper activation of the pathway.
This paper describes the molecular cloning and functional analysis of the EMA-specific gene groovin (grv),
which is identical to kakapo (Prout et al., 1997). kakapo
(kak) has been previously identified in a genetic screen for
mutations that cause wing blisters. This phenotype results
from impaired adhesion between the dorsal and ventral
wing epithelia (Prout et al., 1997
). Kak is a large intracellular protein, expressed mainly along the EMA cell plasma membrane. The primary sequence of Kak exhibits sequence motifs that indicate its possible association with
the cytoskeleton of the EMA cell. Analysis of kak mutant
embryos shows that kak is essential for proper completion
of the muscle-dependent tendon cell differentiation program. Our results suggest that the primary role of Kak is to
mediate the restricted localization and accumulation of
Vein protein at the muscle-tendon junctional site.
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Materials and Methods |
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Fly Strains
Fly stocks used were y w (used as a "wild-type" strain). Df(2R)MK1 = Df
(2R) 50B3-5;50D1-4 was provided by V. Harternstein (University of California, Los Angeles, CA). b pr cn bw was provided by J. Campos-Ortega
(University of Köln, Germany) and was isogenized in our lab. P{ry+17.2 = neo-FRT} 42D; ry605 (referred to as FRT42D in this paper) was provided
by the Bloomington stock center (Bloomington, IN). FRT42DV104/SM5
(referred to as V104 or kakV104 in this paper) was provided by N. Brown
(Cambridge University, UK; Walsh and Brown, 1998). Seven alleles of
kak (Prout et al., 1997
) were obtained from J. Fristrom and M. Prout
(University of California, Berkeley, CA). kakA405 was generated in our lab
by repetitive EMS mutagenesis (screen for lethal mutations over
Df(2R)MK1; see below).
EMS mutagenesis was performed essentially as described in Girgliatti
(1986). Isogenized b pr cn bw males were EMS mutagenized and their F1
male progeny were tested for lethality over Df(2R)MK1. kakA405 was generated in this screen.
Immunochemical Reagents and DNA Probes for Whole Mount In Situ Hybridization
Primary antibodies: anti-Groovin monoclonal antibody (mAb#19) was
raised in mice immunized with Drosophila embryonic membrane proteins
(>100 kD) (Volk and VijayRaghavan, 1994). Hybridomas were screened
by immunostaining and selected according to their staining pattern. Although mAb#19 reacts specifically with the Kak COOH terminus protein
sequence (as shown in Results), we found that it cross-reacts with additional, Kak-unrelated epitope(s) present along the embryonic segmental
grooves. Polyclonal anti-Kak antibodies were generated in guinea pigs by
immunizing with a Kak-glutathione-S-transferase (GST) fusion protein
(containing the Kak sequence encoded by a 4.6-kbp fragment of kak
cDNA [position 6394-10993, encoding amino acids 2136-3668]). Polyclonal anti-Stripe (Becker et al., 1997
), anti-Vein, and anti-Delilah antibodies were prepared in our lab (Yarnitzky et al., 1997
).
Rabbit anti-myosin heavy chain antibodies were obtained from P. Fisher (SUNY, Stony Brook, NY). Secondary antibodies used included: Cy3-conjugated anti-guinea pig and anti-rat fluorescein-conjugated anti- rabbit, and HRP-conjugated anti-rabbit and anti-mouse IgM antibodies (Jackson ImmunoResearch, West Grove, PA).
Nonradioactive digoxygenin-labeled DNA probes for various kak
cDNA fragments and a 1 tubulin fragment (D. Buttgereit, University of
Marburg, Germany) were used for whole embryo in situ hybridization.
Histochemical Staining
For sectioning, embryos were first stained with anti-Kak polyclonal antibody and then embedded in JB-4 embedding media (Polysciences, Inc.,
Warrington, PA) as described (Volk, 1992). Sections (3-4-µm width) were
examined under a Zeiss Axioscope microscope (Thornwood, NY).
Whole Mount Embryonic Staining
Antibody staining was performed essentially as described previously
(Ashburner, 1989), except that the embryos were fixed with 3% paraformaldehyde. In situ hybridization was performed by the method of Tautz
and Pfeifle (1989)
using a digoxygenin-labeled DNA probe.
Western Analysis
Western analysis was performed according to standard procedures and is
described in Volk (1992). SuperSignal chemiluminescent substrate (Pierce
Chemical Co., Rockford, IL) was used for signal detection.
For Western blot analysis of single embryos, kakV104/+ flies were produced and crossed with each other. The embryos of this cross were incubated for 25 h in 25°C, and each of the nonhatched embryos was boiled in sample buffer and subjected to SDS-PAGE in a single lane. The truncated band (Fig. 4 D) is representative of seven individual embryos.
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Expression of FLAG-tagged Kakapo
The kak cDNA insert was amplified by PCR from phage 43d and subcloned in pECE-FLAG (SmaI-XbaI). 293 cultured human cells were
transfected with 20 µg of the above construct by the standard calcium
phosphate transfection method. Transfected and nontransfected cells
were harvested 48 h after transfection and lysed, and the resulting protein
extracts were immunoprecipitated with anti-FLAG antibodies coupled to
agarose beads (anti-FLAG M2 affinity gel; Kodak No. IB13021). Immunoprecipitated proteins were eluted in sample buffer and resolved on
7.5% SDS-PAGE, followed by Western analysis using mAb#19.
Preparation of Fly Genomic DNA and Southern Analysis
FRT42DV104/SM5 flies were crossed to b pr cn bw flies to out-cross SM5. Genomic fly DNA was prepared from FRT42DV104/b pr cn bw progeny of the above cross and from the FRT42D and b pr cn bw lines, according to standard procedure. The genomic DNA was subjected to Southern analysis using genomic probes. The probes were amplified by PCR using Taq DNA polymerase (D1806; Sigma Chemical Co., St. Louis, MO) on genomic DNA of homozygous FRT42D flies. The following primers were used (see also Fig. 4 A): primer A: 5'-CTGTTATGGTGCGCGTGG-3'; primer B: 5'-CGGGTTCGGTTTATCAAG-3'; primer C: 5'-CACTAACATAGAGCTACG-3'; and primer D: 5'-CAGTTGTTGTTGGATGAC-3'.
PCR Analysis on Individual Mutant Embryos
Genomic DNA from individual embryos was prepared as described by
Rastelli et al. (1993). kakV104/SM6B, Cy, Roi, al, dp, cn, sp, P[ry+, eve-lacZ] embryos were collected, aged, permeabilized in heptane/PBS mixture, rinsed, and stained for X-gal for 1.5 h. White embryos were selected,
squashed in Gloor and Engel's buffer (10 mM Tris, pH 8.2, 1 mM EDTA,
25 mM NaCl, 200 µg/ml proteinase K), and incubated at 37°C for 30 min
and at 95°C for 2 min. For each PCR reaction, 1-2 µl of this lysate was used.
Cloning of groovin/kakapo cDNA and Molecular Analysis
Anti-Grv monoclonal antibody (mAb#19) was used to screen a Drosophila embryonic (9-12 h) expression library cloned in gt11 (Zinn et al.,
1988
). A single clone (
43d) was isolated, and it exhibited a similar pattern of expression, by in situ hybridization, as that revealed by the
mAb#19 staining. A total of 12,989 bp of kak cDNA sequence was isolated by successive screens with 5'-end fragments from isolated clones
(
9-1-1,
4,
6,
6P, and
3). The most 5' cDNA sequences (1-3, 13, 700PCR, and 800V) were isolated by PCR amplification from the phage
expression library. All clones and amplified fragments were tested by in
situ hybridization, and they exhibited an EMA cell-specific expression
pattern.
DNA fragments subcloned in Bluescript (Stratagene, La Jolla, CA),
gt11 DNA, or PCR products were sequenced by an automated sequencer (Applied Biosystems, Foster City, CA). The sequences were analyzed by AutoAssembler DNA sequence assembly software package (version 1.0.3; Applied Biosystems).
For Northern analysis, a kak cDNA 4.6-kbp (position 6394-10993) probe was used.
Sequence and Computer Analysis
The predicted amino acid sequence was analyzed by the Wisconsin Package Version 9.1 (Genetics Computer Group [GCG], Madison, WI). Nonredundant databases were searched against the Kak amino acid sequence.
The results of BLASTP 1.4.11 (Altschul et al., 1990), which are representative of other results obtained, have indicated homology to the dystrophin protein family. The region of Kak internal repeats was identified using
COMPARE and DOTPLOT, with the parameters window length = 100, stringency = 39-45 points (not shown). The repeats were aligned using
PILEUP, and the output file was further processed using the SeqVu 1.0.1 program (The Garavan Institute, Sydney, Australia). The repeat border
definition is in line with the dystrophin consensus CS1 (Koenig and
Kunkel, 1990
). The Kak consensus repeat contains a similar amino acid content and order as the dystrophin consensus CS1 (46% identity and
51% similarity). The putative Leucine-zipper domains were identified by
the MOTIFS program of GCG. The results of BLASTP 1.4.11 also indicated that Kak COOH terminus shares a homology domain with additional proteins in the database (Fig. 2 B). The proteins that share this
homology domain with Kak COOH terminus are human brain mRNA
(KIAA0465 protein; sequence data available from GenBank/EMBL/
DDBJ under accession number AB007934), human GAR22 (accession
number Y07846), mouse Gas2 (accession number M21828), human Gas2
(accession number U95032), and a putative open reading frame (ORF)
from Caenorhabditis elegans (Wilson et al., 1994
; accession number
U40800; gi1065956).
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Results |
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Cloning of groovin/kakapo
We have previously identified a monoclonal antibody
(mAb#19), which stained the EMA cells in a specific manner, and according to its prominent staining along the embryonic segmental grooves, we named the corresponding
antigen Groovin (Volk and VijayRaghavan, 1994). To
gain an insight into the molecular nature of Groovin, we
used mAb#19 to screen an embryonic cDNA expression library. Based on this screen, we isolated a single reactive
phage clone (
43d). In situ hybridization with the isolated
43d cDNA insert exhibits embryonic expression in the
EMA cells (similar to the expression pattern obtained
with the monoclonal antibody). The specific reactivity of
mAb#19 with the expressed protein sequence of 43d
cDNA was verified by the following experiment. The
cDNA insert from
43d was subcloned into a FLAG-tag-
containing vector to enable transfection of human cultured 293 cells. After transfection with the 43d-containing
construct, cells were lysed, immunoprecipitated with anti-FLAG antibody, and subjected to Western analysis with mAb#19. A specific reactive band of the expected 43-kD
size was detected only in the transfected cells (Fig. 1 A).
The selected cDNA clone was subsequently used to screen
and clone continuous cDNA sequences. As shown below,
we have demonstrated that groovin is the structural gene
for the kakapo (Prout et al., 1997
) locus. We will refer to
this gene as kakapo (kak).
The contiguous nucleotide sequence of partially overlapping cDNA clones spans 12,989 bp and contains a single continuous ORF (positions 1-12453) encoding 4,151 amino acids (Fig. 1 D). The most 5' sequences of the
cDNA, encoding the putative initiator methionine of the
ORF, were cloned by Gregory and Brown (1998). Thus,
the complete cDNA of kak spans 17,420 bp and encodes a
novel protein of 5,497 amino acids of a putative size of 627 kD. All the amino acid positions indicated in this paper refer to the amino acid sequence presented in Fig. 1 D, which
does not include the two alternative NH2-terminal spliced
forms described in Gregory and Brown (1998)
.
In situ hybridization to polytene chromosomes using a 1.4-kbp fragment of kak cDNA (position 11588-12989) indicates that kak maps to a single location on the second chromosome, at position 50C3-6 of the cytological map (not shown). Northern blot analysis using a kak cDNA fragment of 4.6 kbp (position 6394-10993) revealed two transcripts of ~17.6 and ~15.4 kbp (Fig. 1 B). Western blot analysis of protein extracts from embryos (12-20 h) shows a major protein band in the ~400-kD size range (determined by comparing the Kak band to that of laminin A chain, Fig. 1 C). This major band is seen when using polyclonal antibodies raised against the protein product of a 4.6-kbp fragment of kak cDNA (encoding for amino acids 2136-3668) fused to GST. The specificity of the antibody is demonstrated by its failure to react with extracts derived from embryos homozygous for a deficiency (Df(2R)MK1; see Materials and Methods) that uncovers the kak locus (Fig. 1 C). The discrepancy between the calculated protein molecular mass (627 kD) and the 400-kD band obtained in Western analysis may be indicative of posttranslational modifications of the translated sequences.
Kakapo Is a Cytoskeletal-associated Protein
The primary amino acid sequence of Kak reveals several
domains and motifs that show high degrees of similarity to
three distinct vertebrate cytoskeletal-related protein families, namely plakin (Ruhrberg and Watt, 1997), dystrophin
(Koenig et al., 1988
), and Gas2/GAR22 (Schneider et al.,
1988
; Zucman-Rossi et al., 1996
) (Fig. 2, A-C).
The NH2-terminal region of Kak is homologous to
the NH2-terminal domain of members of the plakin family
of cytoskeletal cross-linker proteins, comprising plectin,
BPAG1, and ACF7 (Ruhrberg and Watt, 1997). These
large proteins link actin microfilaments and intermediate filaments to the plasma membrane at specialized attachment sites, called hemidesmosomes. Abnormal function of
various plakin family members leads to skin (e.g., bullous
pemphigous) as well as neurological (e.g., dystonia musculorum) disorders (for reviews see Ruhrberg and Watt,
1997
; Fuchs and Cleveland, 1998
). The region of similarity between Kak and plakin family members includes the actin binding region but does not exhibit similarity to the intermediate filament-associated domain; homologies with
plakin family members are described in more detail in
Gregory and Brown (1998)
.
The central region of Kak (amino acids 408-3574) consists of 22 repeats, 105-113 amino acids long (Figs. 1 D and
2 A). A computerized search has indicated that the central
region of Kak shares sequence similarity (~20% identity)
with spectrin-like repeats present in an array of cytoskeletal-associated proteins, including dystrophin, -actinin, and
spectrin (Davison and Critchley, 1988
). These repeats are
predicted to adopt a triple-helical conformation (Speicher and Marchesi, 1984
). In dystrophin, the multiple repeat
domain functions as a spacer between the NH2-terminal
actin-binding domain and the COOH-terminal domain associated with a group of membrane proteins (Ervasti and
Campbell, 1991
). The consensus sequence deduced from
the alignment of the spectrin-like repeats in Kak shares
46% identity (51% similarity) with the human dystrophin repeat consensus, CS1 (Koenig and Kunkel, 1990
) (Fig. 2
A). This similarity suggests the presence of a similar domain containing multiple spectrin-like, triple-helical repeats in the central region of Kak protein. This region also
contains five Leucine-zipper motifs (Figs. 1 D and 2 C).
A somewhat lower degree of similarity between the Kak
COOH-terminal domain (sequence 3725-3793) and the
region in dystrophin containing the two EF-hand motifs is
also observed (not shown). The COOH-terminal domain
of dystrophin-related proteins is highly conserved (Roberts and Bobrow, 1998) and includes a WW domain, implicated in mediating interactions with the transmembrane
protein,
-dystroglycan (Jung et al., 1995
). This domain
also includes two putative Ca2+-binding EF-hands (Kawasaki and Kretsinger, 1995
) and a region involved in the
binding to members of the syntrophin family of PDZ domain-containing proteins (Ahn and Kunkel, 1995
; Suzuki
et al., 1995
). The similarity between Kak and dystrophin in
the COOH-terminal domain is detected only along the
EF-hand motifs (~30% identity along a sequence of 70 amino acids). This limited similarity may suggest that although Kak does not appear to be a dystrophin family member, these genes may share a common ancestor.
The COOH-terminal region of Kak shows sequence
conservation with yet another family of cytoskeletal-related proteins representative by the Gas2/GAR22 proteins (Schneider et al., 1988; Zucman-Rossi et al., 1996
)
(Fig. 2 B). Mouse Gas2, a member of this protein family,
belongs to a set of proteins that was shown to be selectively expressed in growth-arrested cells in culture (Schneider et al., 1988
). It is a highly regulated protein (Brancolini
et al., 1992
; Manzow et al., 1996
) that interacts with the microfilament system (Brancolini et al., 1992
; Brancolini and
Schneider, 1994
). Deletion analysis of the Gas2 protein
suggests that the region in Gas2 that is homologous to Kak
has a significant function in cytoskeletal organization
(Brancolini et al., 1995
). During apoptosis, Gas2 is cleaved
by ICE proteases, presumably leading to microfilament derangement (Brancolini et al., 1995
). An additional partially cloned cDNA species of unknown function from human brain (Seki et al., 1997
) exhibits a high level of identity to the COOH-terminal domain of Kak protein (Fig. 2
B). This similarity extends beyond the Gas2 homology domain and exhibits 40% overall identity along the entire
1,658-amino acid sequence available in the data base. It is
not clear whether this partially cloned cDNA represents a
human Kak homologue. In addition, a putative protein
from C. elegans (Wilson et al., 1994
) shares domain contents with Kak, including plectin, dystrophin, and Gas2/
GAR22-like domains mentioned above (see also Gregory
and Brown, 1998
). The function of this putative protein is
yet to be elucidated.
Taken together, the deduced amino acid sequence of Kak (summarized in Fig. 2 C) predicts a novel large intracellular protein that carries two distinct cytoskeletal-associated domains separated by a spacer consisting of elongated triple-helical spectrin-like repeats. At the NH2-terminal domain, Kak may interact with actin microfilaments, while at its COOH terminus, it may be associated with membrane structures or with additional cytoskeletal components. The similarities between Kak and its C. elegans and putative human homologues suggests that Kak structure is conserved through evolution.
Kakapo Marks the Epidermal Muscle Attachment Cells in the Embryo
The expression pattern of kak mRNA and protein during various stages of embryonic development were characterized (Fig. 3). kak mRNA expression is restricted to ectodermally derived cells, and is initially observed in extended germ band embryos during late stage 11. During germ band retraction, the mRNA is expressed by cells along the future segmental grooves and in a small cluster of ectodermal cells at the middle of each hemi-segment (Fig. 3, A-D). Since at these stages the somatic myotubes have not yet extended their leading edge into their final position, we assume that the initial activation of kak transcription is independent of muscle-dependent cues.
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The expression of Kak protein was studied using a polyclonal antibody raised against a bacterially expressed cDNA fragment from the COOH-terminal region. The protein expression pattern of Kak follows its mRNA expression and is apparent from stage 14 of embryonic development. During the final stages of muscle development (e.g., stage 16), high levels of Kak protein are detected in muscle-bound tendon cells, as demonstrated in embryos double-labeled for Kak and myosin (Fig. 3, G-I). Kak protein is concentrated at the circumference of the tendon cell, presumably along the tendon cell plasma membrane (Fig. 3 J). Low levels of Kak protein are observed in other tissues, including epidermal cells and chordotonal organs (not shown).
The Isolation of Mutations in the kakapo Locus
To gain a functional insight as to the role of Kak during
embryonic development, we screened for EMS-induced
mutations that are lethal when crossed with Df(2R)MK1,
a deletion that uncovers the kak locus. We recovered 15 lethal mutations that fall into several complementation
groups. One allele designated A405 failed to complement
a collection of seven alleles of the kak gene (Prout et al.,
1997). A genetic screen (Walsh and Brown, 1998
), similar to that of Prout et al. (1997)
, identified a complementation
group that proved to be allelic to kak. A representative allele from this complementation group, V104, failed to
complement the A405 allele. We concluded that A405 is
allelic to kak.
We performed molecular analysis of kakV104 genomic DNA and identified a chromosomal rearrangement in the 3' end region of the kak gene (see Fig. 4). We carried out Southern analysis with an array of probes covering the entire kak cDNA sequence and identified a region in the COOH terminus of the gene that exhibited a distinct pattern of restriction fragments. The corresponding genomic region was sequenced from the parental (FRT42D) genomic DNA. To further narrow down the region containing the chromosomal rearrangement, we used smaller genomic sequences as probes and tested their potential to recognize the rearrangement. We identified two sequential genomic sequences (AB and CD; see map in Fig. 4 A) that detect a rearrangement in kakV104 mutant chromosome in Southern analysis but react with distinct restriction fragments in the rearranged chromosome (Fig. 4 B). Thus, the rearrangement in kakV104 mutant chromosome is likely to be introduced in the genomic region that includes both fragments (see map in Fig. 4 A).
To further characterize the rearrangement in kakV104, we performed PCR, followed by sequence analysis of individual homozygous kakV104 embryos in the suspected rearranged genomic region (see Materials and Methods for details). Although the size and sequence of the AB and CD genomic fragments was not altered in the mutant embryos, we were unable to amplify the 2.8-kbp AD fragment, which includes both fragments and an additional 400-nucleotide fragment in between (Fig. 4 C). This result together with the Southern analysis (described above) strongly argues that the x ray-induced rearrangement falls into the 400-nucleotide stretch between the AB and CD fragments (Fig. 4 A, hatched box).
The rearrangement at the kak 3' end region in kakV104 chromosome predicts an alteration within the last 280 amino acids of the mutant Kak protein. Indeed, Western analysis of individual homozygous kakV104 embryos with the anti-Kak polyclonal antibody reveals a truncated protein that runs slightly faster compared with the wild-type protein (Fig. 4 D). Thus, it is suggestive that the alteration in kak 3' end region leads to premature translational termination.
Taken together, the Southern, PCR, and Western analyses are consistent with the notion that a DNA rearrangement in the kak gene was introduced in the kakV104
chromosome. Molecular analysis described in the accompanying paper (Gregory and Brown, 1998) shows that in
kak P-element mutant allele (l(2)k03010), the P-element is
inserted at the 5' intronic sequences of the kak gene. Our
molecular analyses and the indication that kakV104 is allelic
to kakl(2)k03010 mutant strongly argue that these mutations
affect the kak gene.
The EMA Cells in kak Mutant Embryos Fail to Differentiate Properly
To elucidate the function of kak in EMA cell differentiation, we analyzed embryos transheterozygous for kakV104/
kakA405 alleles for expression of various markers characteristic of tendon cell terminal differentiation, including
Stripe, Delilah, and 1 tubulin mRNA. This analysis shows
that an excess of EMA cells, marked by the expression of
Stripe and Delilah, is observed at a number of sites in the
epidermis (Fig. 5). This phenotype is particularly notable
in domains in which a group of muscles extend together
towards neighboring epidermal attachment cells, such as
along the ventral segmental border cells, to which the four ventral longitudinal muscles bind (Fig. 5 B, arrow). The
excess in EMA cells was also observed in additional kak
allelic combinations, e.g., embryos transheterozygous for
kakA405/DfMK1. The EMS-induced kakA405 allele appears
to represent a severe kakapo allele. Although the size of
Kak protein is not altered, as concluded from Western analysis of individual kakA405 homozygous mutant embryos (see Fig. 4 D), their mutant phenotype is quite
similar to that of kakA405/DfMK1 mutant embryos (not
shown).
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To further study the state of differentiation of the EMA
cells in the kak mutant embryos, we analyzed the expression of the 1 tubulin gene. In wild-type embryos, the expression of the
1 tubulin gene is significantly elevated towards the end of tendon cell differentiation. In contrast to
the expression of Stripe and Delilah, the mRNA expression of
1 tubulin in kak mutant embryos is significantly
reduced, suggesting that transcription of the latter gene requires different levels of signaling (Fig. 5 B). We suspected
that Vein signaling, which is required for terminal differentiation of tendon cells (Yarnitzky et al., 1997
), may be
reduced in the mutant embryos; while there is enough signal to trigger Delilah and Stripe expression, it is not capable of inducing
1 tubulin transcription.
Vein Localization Is Abnormal in kak Mutant Embryos
Previously, we showed that the expression of delilah,
stripe, and 1 tubulin is induced in the epidermal attachment cells as a result of the EGF-receptor pathway activation by the neuregulin-like growth factor, Vein (Yarnitzky
et al., 1997
). Vein protein localization is restricted to the
muscle-tendon junctional site in wild-type embryos. However, in kakV104/kakA405 mutant embryos, Vein protein is
not localized and appears rather diffuse (Fig. 6). This altered pattern of Vein may explain the multiple number of
cells expressing delilah and stripe; since Vein is not strictly
localized at a given muscle-tendon junction site, it apparently weakly activates the EGF-receptor pathway in
neighboring cells as well. We presume that the only cells
that can respond to the ectopic Vein protein are the competent population of EMA cells, defined by the early expression of stripe (see introduction). These cells express
stripe during early developmental stages in a muscle-independent manner and normally lose their stripe expression by stage 16 of embryonic development. When these competent EMA cells receive the muscle-derived Vein signal,
the expression of stripe and delilah is reactivated. It appears
that only this population of cells is capable of responding
to Vein, since the pattern of the ectopic Stripe- or Delilah-expressing cells in the kak mutant embryos resembles
that of the early population of Stripe-expressing cells (not
shown).
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The reduced levels of 1 tubulin mRNA in the mutant
tendon cells may also result from the abnormal pattern of
Vein localization, since lower levels of Vein may not be
sufficient to induce maximal
1 tubulin expression. It
therefore appears that the primary defect in kak mutant
embryos stems from the lack of Vein accumulation at the
muscle-tendon junctional site.
The Somatic Muscle Pattern Is Disrupted in kakapo Mutant Embryos
Finally, we wished to examine whether the abnormal differentiation of the EMA cells in kak mutant embryos is reflected by the pattern of the somatic musculature. kakV104/
kakA405 mutant embryos at stage 16 of embryonic development were labeled with anti-myosin heavy chain antibody
to visualize the somatic muscles, and the muscle pattern
was compared with that of wild-type embryos. A significant disruption of the somatic muscle pattern is observed
in kak mutant embryos (Fig. 7). In many cases, individual myotubes are not oriented correctly, and in some cases the
myotube rounds up. Since Kak cannot be detected in myotubes using our antibodies, it is assumed that the somatic
muscle derangement is secondary to the abnormal differentiation of the EMA cells. A similar phenotype is also
observed in stripe mutant embryos, in which the EMA
cells do not differentiate correctly (Frommer et al., 1996).
The similarity between the stripe and kak muscle phenotype and the reduced
1 tubulin mRNA expression are
consistent with the conclusion that EMA cell differentiation is defective in kak mutants. The correct recognition
between the muscle and the tendon cell is essential for arresting the extension of the myotube and establishment of
the final pattern of somatic musculature (Yarnitzky et al.,
1997
). It appears that the muscle development in kakV104/
kakA405 embryos does not represent a complete loss of
function phenotype since a more severe muscle defect is
observed in kakV104/DfMK1 embryos (not shown).
|
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Discussion |
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Intercellular inductive interactions take place continuously during development and underlie proper organogenesis. In this paper, we describe a novel protein, Kakapo,
that is essential for the inductive interactions between
muscles and their epidermal attachment cells during
Drosophila embryogenesis. The accompanying papers describe additional functions for this protein in epidermal integrity and cytoskeletal arrangement (Gregory and Brown,
1998) and in proper formation of the neuromuscular junction (Prokop et al., 1998
).
Our results are consistent with a model in which Kak is essential for the concentration of Vein at the muscle-tendon junctional site. This localization is important for the accumulation of Vein protein at high levels; such levels are critical to ensure that a single EMA cell responds to the approaching muscle by turning on its differentiation program towards a fully developed tendon cell.
Possible Mechanisms by which Kakapo Exerts Its Function
Kak is an intracellular protein that exhibits two structural
domains associated with cytoskeletal elements: the actin
binding domain at its NH2-terminal and the Gas2 domain
at its COOH-terminal end, spaced by an elongated, multiple spectrin-like repeat domain. How could this intracellular protein affect the localization of Vein at the extracellular matrix surrounding the EMA cell? We consider at least
two possibilities, which are not mutually exclusive. The
first is the association of Kak with the unique cytoskeletal network of the EMA cell, which is critical for its polarized
organization. Tendon cell polarity may be essential for
maintaining the characteristic junctional complexes formed
between the basal surfaces of the EMA cell and the muscle cells. The space between these junctional complexes
contains many extracellular matrix proteins, some of which may posses a Vein binding function. Impaired tendon cell polarity may lead to the loss of the putative Vein-binding component(s). Alternatively, Kak may be associated with a transmembrane protein(s) responsible for
Vein localization either by direct binding or by association
with additional extracellular matrix components that may
directly bind Vein. Immunoprecipitation experiments with anti-Kak antibody (Strumpf, D., and T. Volk, unpublished) indicated that Kakapo forms protein complexes
containing the extracellular protein Tiggrin (Fogerty et al.,
1994). These results favor the latter possibility that Kak is
directly associated with protein complexes that may be important for Vein binding. The reduced amount of electron-dense material observed at the muscle-tendon junction
site in the kak mutant embryos described in Prokop et al.
(1998)
is in line with both mechanisms mentioned above.
The excess number of Stripe- and Delilah-expressing
cells in the kak mutant embryos may be attributed to the
dispersed levels of Vein, which could induce partial activation of the EGF-receptor signaling pathway in neighboring cells. An alternative explanation is that muscle-dependent differentiation of tendon cells may be accompanied
by lateral inhibition of neighboring cells. The differentiated tendon cell may activate the Notch-signaling pathway
in the surrounding cells. Aberrant contacts between tendon cells and their neighboring EMA competent cells in
the kak mutant embryos may prevent efficient lateral inhibition, resulting in an excess of Stripe- and Delilah-expressing cells. An observation that supports this possibility is
that an excess in 1 tubulin-expressing cells is detected in
Delta mutant embryos (Volk, T., unpublished). Delta, a
well-characterized Notch ligand, mediates lateral inhibition in a large array of tissues during embryonic and adult
development (Artavanis-Tsakonas et al., 1995
). The lack
of Delta may prevent lateral inhibition of the competent
EMA cells, leading to their differentiation into
1 tubulin-
expressing cells. The impaired integrity of the epidermis described by Gregory and Brown (1998)
is consistent with
this explanation.
Kak May Be Part of a Regulatory Mechanism That Controls Vein Activity
The possibility that Kak directly mediates a Vein-induced
signaling cascade was excluded since ectopic expression of
Vein in kak mutants can activate delilah gene expression
in epidermal cells, as in wild-type embryos (not shown).
However, in this experiment the embryos expressing ectopic Vein are exposed to an excess of Vein protein compared with the physiological conditions. High levels of
Vein may compensate for a mechanism responsible for accumulation of Vein at specific sites. Recent findings
(Schnepp et al., 1998; Yarnitzky et al., 1998
) suggest that
Vein is a relatively weak activator of the EGF-receptor
signaling pathway, compared with Spitz, the TGF
Drosophila homologue. Thus, Vein-mediated signaling may
be susceptible to an as-of-yet-uncharacterized regulatory mechanism that controls its localization and accumulation
essential for proper receptor activation. Kak may be part
of such a regulatory mechanism.
The Function of Kak in the Wing May Be Comparable with Its Function in Muscle-dependent Tendon Cell Differentiation
The kak alleles were initially isolated on the basis of their
ability to induce a wing blistering phenotype (Prout et al., 1997). The blistering phenotype is explained by defects in
adhesion between the dorsal and ventral aspects of the
wing epithelia (Prout et al., 1997
; Fristrom and Fristrom,
1993
). The failure of kak mutant cells to adhere to wild-type counterpart cells in the wing may result from the impaired differentiation of the mutant cells. Interestingly,
Vein mRNA is expressed at high levels in the wing intervein domain after puparium formation (Simcox et al.,
1996
). This domain includes the cells that adhere to each
other at the pupae stage to form the adult wing. The role
of Vein in this process is yet to be elucidated. By analogy
to its involvement in mediating Vein localization in the
embryo during the process of muscle-dependent differentiation, Kak may contribute to the correct localization of
Vein protein at the junction formed between the dorsal
and ventral intervein epithelia. This localization may be
essential for the correct activation of the EGF-receptor
pathway and for the proper differentiation of the wing epithelial cells.
Possible Kak-like Activity in Vertebrate Neuromuscular Junction Formation
Several lines of evidence suggest that the process of Vein-mediated tendon cell differentiation shares molecular
similarities with neuromuscular synapse formation. The
secretion of Vein by the approaching muscle cell, and its
restricted localization at the muscle-tendon junctional
site, is reminiscent of the specific localization of its mammalian homologue Neuregulin (NRG or ARIA), which is
secreted from the motor nerve endings and is localized at
postsynaptic sites. NRG is essential for the activation of synapse-specific gene transcription in the postsynaptic
muscle nuclei (Falls et al., 1993; Fischbach and Rosen,
1997
; Burden, 1998
). In a similar manner, Vein is required
to positively regulate the transcription of tendon-specific
genes, such as delilah and
1 tubulin (Yarnitzky et al.,
1997
).
The aberrant formation of the Drosophila neuromuscular junction in kak mutant embryos (described by Prokop
et al., 1998) is consistent with an important role for this
gene in both Vein-mediated tendon cell differentiation
and neuromuscular junction formation, supporting a molecular correlation between these processes.
The mechanism that is responsible for the restricted localization of NRG together with its erbB receptors is yet
to be elucidated. However, when this localization is defective, the structure and proper function of neuromuscular
synapses is disrupted (Sandrock et al., 1997). Thus, vertebrate Kak-like proteins may be part of an intracellular
mechanism that is essential for restricting synaptic sites to
specific muscle-subcellular domains.
![]() |
Footnotes |
---|
Received for publication 24 April 1998 and in revised form 21 October 1998.
This research was supported by a grant from the Israel Science Foundation (to T. Volk).
Address all correspondence to Talila Volk, Department of Molecular Genetics, The Weizmann Institute of Science, 76100 Rehovot, Israel. Tel.:
972-8-9342426. Fax: 972-8-9344108. E-mail: lgvolk{at}wiccmail.weizmann.ac.il
We would like to thank P. Fisher and D. Buttgereit for antibody and cDNA reagents; N. Brown, J. Fristrom, M. Prout, V. Hartenstein, and A. Brand for fly strains; N. Brown, S. Gregory, and A. Prokop for sharing unpublished data; E. Schejter, B. Shilo, and S. Schwarzbaum for critical reading of the manuscript and helpful comments; Li Min, Michal Renert, and Inbal Ben-Zvi Zahor for technical assistance; and Yael Rosenberg-Hasson, Naomi Levy-Strumpf, Tali Yarnitzky, and Shirly Becker for their comments and suggestions.
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Abbreviations used in this paper |
---|
EMA, epidermal muscle attachment; kak, kakapo; ORF, open reading frame.
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