* Friedrich Miescher-Institut, CH-4002 Basel, Switzerland; § Department of Cell and Molecular Biology, Lund University,
S-22100 Lund, Sweden; and Department of Biology, University of Rochester, Rochester, New York 14627
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report the molecular and functional characterization of a new chain of laminin in Drosophila.
The new laminin chain appears to be the Drosophila
counterpart of both vertebrate
2 (also called merosin)
and
1 chains, with a slightly higher degree of homology to
2, suggesting that this chain is an ancestral version of both
1 and
2 chains. During embryogenesis,
the protein is associated with basement membranes of
the digestive system and muscle attachment sites, and
during larval stage it is found in a specific pattern in
wing and eye discs. The gene is assigned to a locus
called wing blister (wb), which is essential for embryonic viability. Embryonic phenotypes include twisted
germbands and fewer pericardial cells, resulting in gaps
in the presumptive heart and tracheal trunks, and myotubes detached from their target muscle attachment sites. Most phenotypes are in common with those observed in Drosophila laminin
3, 5 mutant embryos and
many are in common with those observed in integrin
mutations. Adult phenotypes show blisters in the wings
in viable allelic combinations, similar to phenotypes observed in integrin genes. Mutation analysis in the eye
demonstrates a function in rhabdomere organization.
In summary, this new laminin
chain is essential for
embryonic viability and is involved in processes requiring cell migration and cell adhesion.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LAMININS are large extracellular matrix (ECM)1 molecules usually associated with basement membranes
(BMs), and represent a family of molecules important for development, adhesion, and cell migration (reviewed by Timpl and Brown, 1996). Laminin was initially isolated from tumor cells as a heterotrimer composed of
1,
1, and
1 chains (Chung et al., 1979
; Timpl et al.,
1979
; see Fig. 2). All laminin chains are composed of a series of protein modules that occur in other ECM molecules (e.g., EGF repeats or laminin G domains; see Fig. 2
A). The size of laminin chains is usually >200 kD. Vertebrate studies have revealed the presence of at least five
chains, three
chains, and three
chains that can assemble in a combinatorial manner to form native laminin molecules. All are classified using a recent nomenclature
(Burgeson et al., 1994
). Data so far show that only
,
,
and
heterotrimers are sufficiently stable to be secreted
(Yurchenko et al., 1997), an issue that becomes particularly important when one of the subunits is lacking or mutated due to a genetic defect.
|
Thin but extended sheets of BM require continuous molecular structures which can extend over long distances,
e.g., in blood vessels. BMs are usually thought to provide
sufficient mechanical stability to resist high shearing forces
at the dermal-epidermal junction or to resist hydrostatic
pressure in glomerular loops in the kidney. On the other
hand, BM needs to be flexible, i.e., to respond to rapid
changes in volume in blood capillaries. The major contribution to these properties comes from two networks formed independently from laminins and collagen IV.
Laminin undergoes a thermally reversible polymerization,
and electron micrographs suggest that peripheral short
and long arm interactions are involved in this assembly
(Yurchenco and Cheng, 1993). Additional molecules are
known to interact with laminin, i.e., nidogen, which is
thought to cross-link the laminin and the collagen IV network, or perlecan, a proteoglycan (reviewed by Timpl and
Brown, 1996
).
Different laminin isoforms are not always expressed at
the same site and time. A careful examination of the occurrence in vertebrate embryonic and adult tissues of all chains shows that laminin
chains have distinct expression patterns, with
4 and
5 showing the broadest, and
1
the most restricted expression (Miner et al., 1997
). Moreover, each BM examined contains at least one
chain, but
the composition of
chains within the BMs changed constantly during embryonic development, as assayed in the
kidney (Miner et al., 1997
).
Few data are known about the developmental function
of laminins, mainly because few laminin mutations have
been identified to date. However, mutations in the 2
chain of human laminin have been linked to congenital
muscular dystrophy (Helbling-Leclerc et al., 1995
), and
the classic dy mutation in mouse could also be linked to
defects in the murine
2 chain (Xu et al., 1994
). In both
species, the lack or partial loss of function of laminin
2
leads to variation in skeletal muscle fibers and muscle fiber necrosis. These findings demonstrate a role for the
2
chain in skeletal muscle function. Mutations in the
2 subunit of laminin can lead to Herlitz's junctional epidermolysis bullosa (Aberdam et al., 1994
; Pulkkinnen et al., 1994),
characterized by blister formation within the dermal-epidermal BMs. Furthermore, mutations in the
3 and
3
laminin chain which associate with
2 to form laminin 5 show similar phenotypes (Kivirikko et al., 1995
; Cserhalmi-Friedman et al., 1998
). Laminin
2 also plays a role
in molecular pathogenesis of neural tropism since the bacterium Mycobacterium leprae binds to
2 on Schwann cell
axon units (Rambukkana et al., 1997
).
Intensive studies on the composition of the ECM in invertebrates have shown the existence of a laminin with a
proposed subunit composition 3, 5;
1;
1 (Montell and
Goodman, 1988
, 1989
; Chi and Hui, 1989
; Kusche-Gullberg et al., 1992
; Henchcliffe et al., 1993
).
3, 5 was previously called lamA, and this new name is proposed as a reminder that
3, 5 is the precursor of both vertebrate
3
and
5 chains. Genetic studies have shown that null mutations in the Drosophila
3, 5 chain lead to embryonic lethality, with visible defects in mesodermally derived tissues, i.e., in heart, muscles, or gut leading to dissociated
cell groups in the various organs (Yarnitzki and Volk,
1995). These data suggest that laminin is used to confer
structural support and adhesivity. Surprisingly, no obvious
pathfinding defects in central nervous system neurons were observed during embryogenesis (Henchcliffe et al.,
1993
), however, at the neuromuscular junction, the extent
between neuronal and muscular surfaces appeared significantly altered in
3, 5 mutants (Prokop et al., 1998
).
Hypomorphic mutants and heteroallelic mutant combinations of 3, 5 can give rise to viable pupae and some viable adults (Henchcliffe et al., 1993
). These adult escapers
show abnormalities in the shape of their legs and in the organization of ommatidia in the compound eye (Henchcliffe et al., 1993
). A recent report has also shown the requirement of
3, 5 in normal pathfinding by ocellar pioneer axons (Garcia-Alonso et al., 1996
).
In spite of the observed pleiotropy of mutations in the
3, 5 gene, the phenotypic effects seen in mutant animals
are not dramatic given the wide distribution of the protein.
This predicted the existence of a second laminin
chain
which can compensate for loss of
3, 5 function. Indeed,
during the course of the Drosophila genome sequencing
program, we noticed the presence of sequences related to
laminin, and subsequent analysis of the genomic region allowed us to define a new member of the invertebrate laminin
chain family, similar to both the vertebrate
1 and
2 chain. We show that mutations from the wing blister
(wb) locus are associated with lesions in this new
gene
and that this second Drosophila laminin
chain is indispensable for embryonic viability and adhesiveness between cell layers.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fly Stocks
The wb alleles, wbk05612, wbk00305, wbPZ09437, wbPZ10002, wbSF25, wbHG10, and
wbCR4, were used to determine embryonic functions for the Wb protein. P
element induced alleles, wbk05612, wbk00305, wbPZ09437, and wbPZ10002, were
produced in the laboratories of Istvan Kiss and A. Spradling (Carnegie Institute, Baltimore, MD), and ethylmethane sulfonate (EMS) induced alleles, wbSF25 and wbHG10, were produced in the laboratory of M. Ashburner (University of Cambridge, Cambridge, UK). wbSF25, wbHG10, and
wbPZ09437 have been described previously (Karpen and Spradling, 1992;
Lindsley and Zimm, 1992
). Df(2L)fn7 (breakpoints 34E3; 35B3-4) and
Df(2L)fn36 (breakpoints 34F3-5; 35B4) were used in this study and are described in Lindsley and Zimm, (1992). Revertants of wbPZ09437 were obtained by precise excision of the P element and showed wild-type appearance and fertility. Lethal chromosomes used in this study were kept in
stocks balanced over CyO (Lindsley and Zimm, 1992
).
Somatic clones in the eye were produced by inducing mitotic recombination using the FLP/FRT system as described in Roote and Zusman (1996).
Videomicroscopy
Embryos from mutant lines were placed on petri perm plates (Hereus) in
a drop of Voltalef 3S oil. All embryos were derived from mothers homozygous for the klarsicht (kls) mutation, which clears out yolk and
makes embryonic phenotypes easily visible during filming, yet has no discernible effect on embryonic development (Wieschaus and Nüsslein-Volhard, 1986). Time lapse videomicroscopy was performed on embryos under a Zeiss Axioskop microscope with a Panasonic AG-6730 recorder and
a Zeiss ZVS-47N CCD videocamera system. wb embryos were identified
by their inability to hatch and the presence of a dorsal hole at the end of
embryonic development.
Immunostaining and Preparation of Embryos for Whole Mounts
Embryos were collected on agar/apple juice plates and prepared for immunostaining according to the protocol described in Zusman et al. (1990)
with an antibody against a pericardial protein (Mab#3; Yarnitzky and
Volk, 1995
) or an antibody against a tracheal protein (2A12; Samakovlis
et al., 1996
). Embryos stained with antibodies were dehydrated and
mounted in a 3:1 solution of methyl salicylate and Canada balsam for examination under bright-field illumination.
For examination of somatic muscles, wb embryos were prepared as described by Drysdale et al. (1993) and viewed under polarized light. To
confirm and examine further the wb somatic muscle phenotype, embryos
derived from parents heterozygous for wb were stained with antibodies
against muscle myosin (Kiehart and Feghali, 1986
) using the procedures
described in Young et al. (1991) and Roote and Zusman (1995)
.
Late stage wb or deficiency-containing embryos were identified by the dorsal hole phenotype and/or their inability to hatch. At earlier stages wb phenotypes were based on 25% of the population exhibiting defects not observed in a wild-type population, and the similarity of these defects to those observed when a dorsal hole is present. The mutant tracheal phenotype was also observed in developing wb embryos using videomicroscopy.
DNA and RNA Techniques
Southern and Northern blot analyses were performed by standard procedures (Maniatis et al., 1982). RNA was extracted by the guanidium thiocyanate/phenol/chloroform extraction method of Chomczynski and Sacchi
(1987)
. Poly(A)+ RNA was isolated using a Pharmacia Kit (Pharmacia
Biotech, Inc.). Equal specific activity of wb probes and laminin
3, 5 and
1 probes were achieved using a standardized labeling protocol, and by
using probes of similar lengths and similar GC content. Exposure times
for Northern blots were 3 d. Whole mount in situ hybridizations were conducted using digoxigenin labeled wb cDNAs following the protocol of
Tautz and Pfeiffle (1989)
.
Verification of the Sequence of Genomic DS Phages
At least three sequence errors were discovered within the published DS
03792 sequence leading to reading frame shifts. Suitable cDNAs were isolated using PCR, subcloned, and were used to correct the derived cDNA
sequence. Irregularities between the domain structure of vertebrate and
this new Drosophila laminin chain were confirmed by additional isolation of suitable cDNAs by PCR and subsequent sequencing, ruling out
misleading interpretations of intron-exon boundaries.
Generation of Antibodies and Staining of Embryos
Two independent fragments from either the NH2 or COOH terminus (amino acids 173-376 and amino acids 2,383-2,633, respectively) were cloned into the appropriate pMALc2 expression vectors (BioRad Laboratories). After induction and lysis of cells, fusion proteins were purified over a maltose matrix (BioRad Laboratories). Both antigens were used to generate two independent rabbit polyclonal antisera each. Polyclonal antisera were affinity purified over a corresponding GST fusion protein (Pharmacia Biotech, Inc.) column and eluted with 0.1 M glycine, pH 2.5. The specificity of the antisera was tested on Df(2L)fn36 embryos. For histochemical staining, the antifusion protein antisera were used at a concentration of 1:500.
Western Blotting
Samples of embryonic extracts and conditioned medium of Schneider S2 cells were separated under nonreducing and reducing conditions on 6% SDS-PAGE. After transfer onto nylon membranes, blots were probed with anti-wb antibodies and detected with HRP conjugated secondary antibodies, followed by ECL chemiluminescence (Nycomed Amersham, Inc.).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning, Sequence Analysis, and Properties of a New Drosophila Laminin Chain
In our attempt to find laminin-like sequences from Drosophila in the database, we noticed the presence of EGF-like repeats similar to laminin chains on the reverse strand
of a subclone derived from the genomic phage DS 03792 (Kimmerly et al., 1996). Subsequent alignment of all subclones derived from this DS phage revealed the presence
of a novel laminin chain gene in Drosophila. Analysis of
the gene structure showed a genomic region spanning ~70
kb of DNA with
16 exons contained within two overlapping DS phages, DS 037092 and DS 01068 (Fig. 1 B). Most
intron-exon boundaries proposed by GENSCAN (Burge
and Karlin, 1997
) were confirmed by isolating and sequencing suitable cDNA clones spanning the region of interest (data not shown).
|
|
Conceptual translation of the 10,101-nucleotide open
reading frame yields a protein of 3,367 amino acids with a
deduced molecular size of ~374 kD (Fig. 2 A). At the NH2
terminus, the predicted initiating methionine is followed
by an amino acid sequence containing structural regions
characteristic of a secretory signal sequence (Fig. 2 A; von
Heijne, 1986). A hydropathy profile of the primary structure revealed no other long hydrophobic regions indicative of a transmembrane spanning segment (Fig. 2 A), suggesting that this laminin chain is a secreted protein.
Closer inspection of the domain structure shows that
this new chain has all the domains of laminin chains in
the appropriate order (Fig. 2 C). However, the number of
different modules varies in some regions. For example, the
second EGF-like stretch contains 10 full and 2 half EGF
repeats, while in vertebrates there are 8 full and 2 half
EGF repeats (Fig. 2 C). In addition, a unique NH2-terminal extension of ~120 amino acids is present (Fig. 2 C). Finally, the array of the second EGF repeat region is symmetrically interrupted by an insertion of 45 amino acids.
We performed domain-wise comparisons of identities to
existing vertebrate chains. The LN domain showed almost an equally high degree of identity to vertebrate
1
and
2 chains, while the LE4 domain showed a slightly
higher degree of identity to vertebrate
2 than to
1.
However, both L4 domains showed slightly higher scores
of identity to
5, immediately followed by equally high
scores to
2 and
1. The remaining two EGF-like repeats
showed that the first was highly homologous to
1 but the
second was homologous to
2. Finally, all five G domains
showed a slightly higher similarity to
2 than to
1. In
summary, the majority of the domains showed most similarity to vertebrate
2 chains, yet many were significantly
similar to
1. For this reason, and to illustrate the fact this
chain is a common precursor of vertebrate
2 and
1
chains, we have tentatively called this chain Drosophila laminin
1, 2 in the remainder of the text.
A special feature within the amino acid sequence should
be noted: the presence of a RGD within the first L4 domain (Fig. 2). RGD tripeptides have been shown to mediate cell adhesion in Drosophila using Drosophila PS2 integrins as receptors (Bunch and Brower, 1992). In fact, a
recent study based on cell culture assays demonstrated that the laminin
1, 2 subunit showed exclusive binding to
one integrin isoform,
PS2m8
PS4A, while the other PS2
integrin isoforms did not show any binding (Graner et al.,
1998
), suggesting that
1, 2 is a ligand of a splice-specific
form of the PS2 integrins.
Temporal and Spatial Distribution of
Laminin 1, 2 Transcripts
Northern analysis was performed on RNA derived from
samples spanning the Drosophila life cycle using 1, 2 cDNAs as probes. An 11-kb transcript was first detected
in the early stages of embryogenesis and peaked in 6-12-h
embryos (Fig. 3 A). In the last part of embryogenesis (12-
18 h), a slightly smaller version of a 10.5-kb transcript was
observed. We cannot exclude the possibility of an alternative spliced transcript or alternative usage of another polyadenylation site. Transcription decays in the later stages and is hardly detectable in third-instar larvae, but increases again in pupal stages. To compare the existing
Drosophila laminin chains, the same Northern blot used
for
1, 2 was also probed with a mixture of
3, 5 and
1
probes (Fig. 3 C). This showed that the two laminin subunits are present at similar stages during embryogenesis.
There is a marked difference, however, as these two subunits are also transcribed very strongly during the late stages of embryogenesis, in contrast to
1, 2 which fades
out rapidly during this stage. Assuming that all probes in
this analysis had similar specific activities (see Materials
and Methods), it suggests that
1, 2 is less abundantly expressed than
3, 5, a feature already noted in vertebrate
expression studies (Miner et al., 1997
).
Using digoxigenin-labeled probes, the spatial expression
of the 1, 2 chain was examined. Transcripts were first detected during oogenesis in nurse cells and growing oocytes
(Fig. 4 A), suggesting a maternal contribution. During
cleavage stage, the message is uniformly distributed in the
egg (Fig. 4 B) and becomes slightly enriched in cells of the
trunk region at blastoderm stage (Fig. 4 C). During germband extension (Fig. 4 D), low levels of uniform expression are observed. After germband retraction, the visceral
mesoderm of the gut starts to accumulate
1, 2 transcripts (Fig. 4, E and F). At that time, cells near the presumptive
muscle attachment sites show transcripts (Fig. 4 F). At
stage 14, strong expression is also observed in cardiac cells
(Fig. 4 G) and more prominent in cells near the muscle attachment sites (Fig. 4, H and I).
|
Transcription of laminin 1, 2 is also readily detectable
in imaginal discs, as assayed by LacZ staining of imaginal
discs derived from the viable P element line H155 which
mimics the embryonic transcript pattern faithfully (data
not shown). Particularly strong expression was found in
wing discs, where certain groups of cells in the presumptive wing dorsal and ventral region show LacZ staining (Fig. 4 J). Strong staining was also observed in the eye antennal disc immediately behind the morphogenetic furrow (Fig. 4 K), and also in a specific pattern in leg discs
(Fig. 4 L).
Spatial Expression of the 1, 2 Protein
To assess the nature and appearance of the 1, 2 protein,
polyclonal antisera against the NH2 and COOH termini
(see Materials and Methods) were produced and assayed
both by Western analyses and on whole mount embryos.
Western blotting of conditioned medium of Schneider S2
cells showed a single 360-kD band (Fig. 3 D, lane 3), while
in embryonic extracts proteolytic cleavage was observed giving rise to a 240-kD band (lanes 4 and 5) and a 110-kD
band (lane 5) which are detectable using anti-NH2 and
-COOH antibodies, respectively. This suggests that proteolytic cleavage also occurs in Drosophila, as was reported for the vertebrate
2 chain (Ehrig et al., 1990
). NH2
antibodies also detected a possible further degradation
product of ~180 kD (lane 4), which is not detected by
COOH antibodies. Both antisera recognize a single 800-kD
band under nonreducing conditions (lanes 1 and 2), suggesting that the
1, 2 protein is part of a laminin trimer.
Using an immunoprecipitation assay,
1, 2 was found to
be associated with the same
and
chains, as was
3, 5 (data not shown).
The protein is first detected at stage 10 as a weak diffusive stripe between the ectoderm and the mesoderm (Fig.
5 A). During germband retraction (Fig. 5 B) the protein is
localized diffusely around areas that constitute the visceral
mesoderm. At stage 14, strong staining is observed in the
BMs that surround the digestive system, i.e., the gut (Fig. 5
C), or at muscle attachment sites (Fig. 5 D, G, and H).
These patterns are strongly reminiscent of the expression
patterns of various Drosophila integrin subunits, particularly the subunit (Leptin et al., 1989
) and the
2 subunit (Bogaert et al., 1987
). Later stages include localization in
dorsal structures along the ventral nerve cord (Fig. 5 E),
and BMs around the digestive system (Fig. 5 F). During
imaginal wing disc development,
1, 2 is localized in a specific spot pattern on the presumptive wing dorsal and ventral region (Fig. 5 I).
|
The wb Gene Encodes Laminin 1, 2
Genomic phage DS 03792 (Fig. 1 B) was mapped to chromosomal region 35A1 (Fig. 1 A). Several P element insertion events could be detected within the genomic area of
the laminin gene. Of particular interest were two fly lines
conferring embryonic lethality that showed the P element
inserted into the middle of the fourth intron (Fig. 1 B). Because insertions of this type showed lethality on other occasions (Horowitz and Berg, 1995) where an unusual splicing event was shown to be the cause for lethality, we
wondered whether the same situation would apply here.
To test whether trans-splicing between the fourth exon of
laminin
1, 2 and the last exon of ribosomal protein S12,
which resides on the P element construct, we performed
Northern analysis on RNA derived from l(2) 09437 embryos or l(2) 10002 embryos (not shown). Two bands were
visible: the doublet band ~11 kb, already detected in the
developmental Northern analysis generated by the wild-type gene from the balancer chromosome, and a smaller
species of 5.6 kb, derived from the mutant chromosome
whose RNA showed trans-splicing to S12, yielding a
shorter transcript (Fig. 3 B). Rehybridization of the same
Northern lane using a S12-specific probe confirmed the
same 5.6-kb mRNA species (data not shown). We interpret the fact that the 5.6-kb mutant band is stronger than
the wild-type 11-kb band as a composite result of a higher
efficiency to complete the transcript, because the mutant
transcript is more stable, or the transfer of larger mRNA is
less efficient. The shortened mRNA codes for a protein truncated within LE8 (Fig. 2 C), and as a result no assembly of the heterotrimeric laminin molecule can occur, as
only
,
, and
heterotrimers are sufficiently stable to be
secreted (Yurchenco et al., 1997
). Consequently, it is likely
that no functional laminin of the subunit composition
1,
2;
1;
1 is secreted in the l(2) 09437 mutant.
A survey in the chromosomal area of 35A showed that a
locus, termed wb, resulting in blisters in wings, could account for the loss of laminin function. To test this hypothesis, l(2) 09437 and l(2) 10002 flies were crossed to suitable
viable and embryonic lethal wb alleles, and were tested for
complementation. None of the strong ethylmethane sulfonate induced wb alleles complemented l(2) 09437 and
l(2) 10002 for embryonic lethality (data not shown). This
result, in combination with the mapping data, strongly argues that l(2) 09437 and l(2) 10002 are mutations in the wb
locus, and that wb is indeed laminin 1, 2.
Defects in wb Embryos
To examine the functions of the Wb protein during embryogenesis, the development of embryos homozygous for
embryonic lethal mutations in the wb gene (wbk05612,
wbk00305, and wbHG10; Lindsley and Zimm, 1992; Zusman,
S., unpublished results) was examined and compared with
wild-type embryos and embryos homozygous for a deficiency that uncovers the wb locus (Df[2L]fn36 and
Df[2L]fn7; Lindsley and Zimm, 1992
).
Time lapse videomicroscopy of developing flies revealed that homozygous wbHG10, wbk05612, and Df(2L)fn36
embryos become abnormal during gastrulation. Rather
than extending their germbands dorsally, mutant germbands twist and extend laterally (Fig. 6, A and B). Near
the completion of germband extension, wb and Df(2L)fn36
embryos show a distinct separation between the mesodermal and ectodermal tissue layers of the germband (Fig. 6,
C and D). These phenotypes are similar to that described
for mys hemizygous embryos which lack the PS subunit of
integrin (Roote and Zusman, 1995
), a potential receptor
for laminin (reviewed by Hynes, 1992
). Another phenotype in common with mys embryos (Wieschaus et al.,
1984
) includes a dorsal hole which often forms in the
cuticle of wbk05612, and occasionally in wbk00305 embryos.
Although Wb protein accumulates around the BMs of the developing embryonic gut, no defects were detected in gut
morphology or midgut primordial migration.
|
Previous studies of embryos lacking the Drosophila
laminin 3, 5 chain have demonstrated functions for this
molecule in the proper morphogenesis of heart, somatic
muscle, and trachea (Yarnitzky and Volk, 1995
; Stark et al.,
1997
). In laminin
3, 5 deficient embryos there is a dissociation of the pericardial cells of the heart, gaps in the dorsal
trunk of the trachea, and the ventral oblique muscles fail to
reach their attachment sites. Similar heart and tracheal defects are found in embryos with mutations affecting
PS3
PS
integrin (Stark et al., 1997
). To determine if the Wb protein is also involved in these processes, we examined the
development of their heart, trachea, and somatic muscles.
The heart (dorsal vessel) forms from external pericardial cells and internal cardioblasts that migrate during dorsal closure to meet along the dorsal midline to form the heart tube (Bate, 1993). wb and wb-deficient embryos stained with antibodies that recognize pericardial cells show that homozygous wbk05612, wbk00305 (both occasionally showing dorsal holes), and Df(2L)fn36 embryos often contain fewer pericardial cells than wild-type embryos resulting in distinct gaps in the heart tube (Fig. 7, A and B). Furthermore, the pericardial cells appear to dissociate randomly and the tube often appears to curve off towards the lateral side of the embryo.
|
The dorsal trunk of the trachea is formed by migration
of the tracheal pits to form a long tracheal tube which extends the length of the embryo (reviewed in Manning and
Krasnow, 1993). Antibodies were used to examine trachea
formation in wb and wb deficient embryos. Embryos homozygous for wbk05612, wbHG10, and Df(2L)fn36 were observed to have significant gaps in the dorsal trunk of the
trachea (Fig. 7, C and D). This was confirmed by examining the development of filmed wb embryos.
Due to the strong expression of the Wb protein in muscle attachment sites, we also examined wb and wb-deficient embryos for defects associated with the attachment of myotubes to their ectodermal attachment sites. Careful examination of somatic muscle in homozygous wbk05612, wbHG10, and Df(2L)fn36 embryos stained with antimyosin antibodies (Fig. 7, E and F), or prepared for examination under polarized light at the end of embryonic development, revealed that their somatic myotubes are often not attached to target epidermal attachment sites. This defect most commonly involves the ventral oblique muscles located in the anterior most segments of the embryo (Fig. 7 F). Random disorganization of myotubes and areas without myotubes are occasionally observed in these embryos as well.
In conclusion, several defects are observed in wb embryos, some in common with those observed in laminin 3,
5 embryos, and many in common with those observed with
integrin mutations.
Defects in Adults
As the name implies, mutations in the wb locus can lead to
blistering of the wing, in which the dorsal and ventral wing
surfaces separate (Woodruff and Ashburner, 1979). As
shown in Fig. 8, A and C, the blisters are located centrally
within the wing, consistent with the location of laminin expression and localization in wing discs (Fig. 4 J and Fig. 5
I). The blisters vary in size, depending on the allelic combination used (data not shown). Homozygous viable alleles of wb exist that show no blistering (i.e., wbCR4), and
only in combination with an embryonic lethal wb allele (i.e., l(2) 09437) or a deficiency (Df[2L]fn7 or fn36) were
blisters observed, suggesting that below a certain threshold, the lack of functional laminin can lead to blistering.
No haplo-insufficiency was observed in l(2) 09437 animals
(data not shown). The wb phenotype strongly resembles
the phenotypes associated with mutations in integrins
(Brower and Jaffe, 1989
; Brabant et al., 1993; Brower et
al., 1995
), and mutations in the Drosophila laminin
3, 5 gene can also lead to blistered wings (Henchcliffe et al.,
1993
).
|
Due to the fact that high expression of wb was also
found posterior to the morphogenetic furrow in the developing eye (Fig. 4 K), we wished to determine the function
of wb during eye development. For this reason, somatic
clones were induced in the eye of wbk05612 flies using the
FLP technique (Golic, 1991). As evident in Fig. 8 D, the
number of photoreceptor cells did not change, but they appear disorganized. Disorganized photoreceptor cells were
also detected in mys and mew (PS1-encoding) mutant
clones (Zusman et al., 1990
; Brower et al., 1995
).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have demonstrated the existence of a second laminin chain in Drosophila, and sequence analysis shows that it is
homologous to the
2 and
1 chain in vertebrates. Most
likely, this chain represents one of the ancestral versions of
a vertebrate
chain of laminin, as some marked changes
are observed in comparison to
1, 2. The protein is slightly
larger than vertebrate
1 or
2, mainly due to the addition
of a NH2-terminal extension, an insertion in the first EGF-like region, and by acquisition of two additional EGF-like
modules (Fig. 2 C). Other discrepancies have been observed in the Caenorhabditis elegans
1, 2 where one G
module is deleted (Fig. 2 C). Laminins have also been isolated in lower organisms such as Hydra vulgaris (Sarras
et al., 1994
) where they are expressed in the subepithelial
zone involved in attachment of mesoderm to the ectoderm. Sequence comparisons suggest that the
chain
associated with this laminin corresponds to an ancestral
version of the
3 and
5 chain (Sarras, M., personal communication).
Virtually no exon boundaries match the gene structure
observed in human laminin 2 or C. elegans laminin
1, 2, nor is the number of exons similar (16 versus 64 and 10, respectively; Zhang et al., 1996
; Fig. 2), suggesting that
chains in higher animals have become more complex by
splitting coding sequences through uptake of new noncoding sequences. In addition, no exon boundaries of Drosophila
1, 2 fit those of Drosophila
3, 5 (Fig. 2 D) or even of C. elegans
1, 2 (Fig. 2 C), suggesting that the two
chains diverged much earlier. Based on the sequenced
C. elegans genome, which discovered only two
chains, it
is plausible to assume that invertebrate genomes such as
Drosophila or C. elegans probably possess only two
chains, one
and one
chain, respectively, which may
limit the number of possible assemblies into functional laminin trimers to two.
A comparison between expression patterns of 1, 2 and
vertebrate laminins reveals that the expression of vertebrate
2 fits better to Drosophila
1, 2, as
1 shows a
highly restricted expression in kidney, as compared with
2 whose expression was reported to be widespread in
mesenchymal cells (Miner et al., 1997
). In accordance with
vertebrate expression studies (Miner et al., 1997
) where
5
was shown to be the most widely expressed
chain,
Drosophila
3, 5 is more widely expressed than
1, 2.
Interestingly, Wb harbors a RGD sequence located on
the L4 domain (Fig. 2 C) which makes it a likely ligand for
integrins. Biochemical studies on integrin-mediated adhesion using Drosophila cell lines identified Wb as a distinct
ligand for PS2m8
PS4A integrin (Graner et al., 1998
),
one of four splice forms of the
PS2
PS integrins (Brown
et al., 1989
; Zusman et al., 1990
). The
PS2 isoform is also
the predominant splice form present at developmental stages during which Wb is expressed (Brown et al., 1989
).
No data have been reported to date on the isoform distribution of
PS integrin. In contrast, other RGD containing
proteins such as tiggrin (Fogerty et al., 1994
), or ten-m
(Baumgartner et al., 1994
) show no absolute requirement
for a specific splice isoform of
PS: both proteins need
only exon 8 of
PS2 to be present. Using a similar approach, Drosophila laminin containing
3, 5 was shown to
be a specific ligand for
PS1
PS integrin (Gotwals et al., 1994
). This suggests that Drosophila laminins (subunit
composition
1, 2;
1;
1, and
3, 5;
1;
1) can serve as
PS2 and PS1 integrin ligands, respectively. Moreover, the
model for embryonic muscle and pupal wing attachment
proposed by Gotwals et al. (1994)
holds true, by juxtaposing another partner to tiggrin facing the PS2 binding site.
Interestingly, the region harboring the RGD in L4 of Wb
is highly related to the RGD-containing site of vertebrate laminin
5 (Graner et al., 1998
), which could indicate that
vertebrate
5 has taken up this motif during evolution, in
contrast to the existing Drosophila
3, 5 which does not
harbor an RGD site. Genetic data further support an association of wb with integrins, since weak mys mutations increase the size and frequency of blisters in wb flies (Khare,
N., and S. Baumgartner, manuscript in preparation). No
conclusive genetic interaction data were reported to occur between
3, 5 and mys (Henchcliffe et al., 1993
).
Several embryonic wb phenotypes (Fig. 6) were shown
to be remarkably similar to those of single integrin mutations, i.e., the separation of mesoderm and ectoderm, and
the twisted germband common to mys (Fig. 6, B and D;
Roote and Zusman, 1995) or to scb (Stark et al., 1997
).
Notably, separated mesoderm/ectoderm and twisted germband were not observed in mutations in the
3, 5 chain
(Yarnitzki and Volk, 1995). The
3, 5 chain was only
found to be required for later stages of patterning of mesodermally derived cells, suggesting that
1, 2 is exclusively
used to confer early adhesion between mesoderm and ectoderm. In contrast, common phenotypes between
1, 2 and
3, 5 were detected in late stages of embryogenesis
where the formation of the ventral oblique muscles is disturbed, particularly in the anterior segments (Fig. 7 F; Yarnitzki and Volk, 1995; Prokop et al., 1998
). Finally, the
formation of the heart was reported to be disturbed in mutations of both genes (Fig. 7 B; Yarnitzki and Volk, 1995).
No phenotype reminiscent of the muscular dystrophy-like phenotype in vertebrates was observed in our mutants. Although we did not observe wb expression in muscles, we cannot rule out marginal expression levels below
the sensitivity of our detection method. However, certain
myotubes do appear disorganized in wb mutant embryos.
This cannot be considered an analogous situation to dy/dy mice (Xu et al., 1994), because the defects observed are
most likely due to the inability of muscle cells to migrate
properly and a failure in attaching to muscle attachment
sites. Similar phenotypes were also observed in laminin
3, 5 mutants (Prokop et al., 1998
).
Previous studies have shown that integrin-mediated adhesivity between the two epithelial cell layers of the wing
is particularly sensitive to mutations involving either integrin ligands (this paper) or upstream factors of integrins,
i.e., the blistered (bs) gene encoding a Drosophila serum
response factor (SRE; Montagne et al., 1996). bs and integrins interact genetically (Fristrom et al., 1994
) and mys
expression appears to be greatly reduced in hypomorphic
bs mutants (Montagne et al., 1996
), suggesting a scenario
where bs might directly control integrin gene expression on the transcriptional level. It is plausible to assume that
bs might also directly control wb expression, as the transcript pattern of both show striking coexpression (Fig. 4 J;
Montagne et al., 1996
), and a corresponding SRE has been
located 260 bp upstream of the putative TATA box of the
wb gene (data not shown).
Specific screens have been performed for mutations affecting adhesion between wing surfaces (Prout et al., 1997;
Walsh and Brown, 1998
). To our surprise, none of the loci
described correspond to wb, suggesting that the formation
of blisters in the wing depends on subtle changes of wb activity. This is further suggested by the fact that only suitable wb allelic combinations show blisters. For example,
blisters were only detected in transheterozygous allelic
combinations of a weak (homozygous viable) allele, wbCR4,
and Df(2L)fn7 or l(2) 09437 which behaves as a null allele.
In other words, only the range of wb activity slightly below
50% of wild-type activity is capable of forming blisters,
while a level of
50% does not affect wing blistering, as
no haplo-insufficiency is observed in l(2) 09437 flies.
In parallel to the wing, wb clones induced in the eye
cause similar phenotypes to clones induced in integrin mutations, i.e., PS1 (mew) mutants (Roote and Zusman,
1995
) or
PS (mys) mutants (Zusman et al., 1990
; Brower
et al., 1995
), but not in
PS2 (if) mutants (Brower et al.,
1995
) which result in virtually wild-type eyes. Similar phenotypes were also observed in laminin
3, 5 mutant combinations, however, the degree of severity of disorganization is higher than in wb or integrin mutant clones (Henchcliffe
et al., 1993
).
![]() |
Footnotes |
---|
Address correspondence to S. Baumgartner, Department of Cell and Molecular Biology, Section for Developmental Biology, Lund University, Box 94, S-22100 Lund, Sweden. Tel.: 0046-46-222-3893. Fax: 0046-46-211-3417. E-mail: stefan.baumgartner{at}medkem.lu.se
Received for publication 2 October 1998 and in revised form 22 February 1999.
Sequence data reported in this paper appears in GenBank/EMBL/DDBJ
under the accession number AF 135118.
D. Martin and S. Zusman contributed equally to this work.
S. Zusman and X. Li's present address is Department of Functional
Genomics, Novartis Pharmaceuticals, Life Sciences Building, 556 Moris
Avenue, Summit, NJ 07901-1398. E.L. Williams' present address is Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77096.
We wish to thank John Roote and Michael Ashburner for their generous contribution of wb alleles, and Denise Montell for providing the H155 line. We also wish to thank Talila Volk, Mark Krasnow, and Dan Kiehart for providing antibodies, and Jürgen Engel for providing mouse EHS laminin. We would like to thank the Berkeley Drosophila Genome Project and particularly Suzanne Lewis for providing sequence information before publication. The help of Herbert Angliker in resequencing portions of the gene is acknowledged. In addition, we appreciate the efforts of Kassandra Gorham, Christopher Wright, Liam Casey, and Mark Hickery on this project.
This work was supported by National Science Foundation grant 9404055 to S. Zusman and by the Schybergs Stiftelse and a Swedish Naturvetenskapliga Forskningradet Grant 409005501-5 to S. Baumgartner.
![]() |
Abbreviations used in this paper |
---|
BM, basement membrane; ECM, extracellular matrix; wb, wing blister.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Aberdam, D., M.F. Galliano, J. Vailly, L. Pulkkinen, J. Bonifas, A.M. Christiano, K. Tryggvason, J. Uitto, E.H. Epstein, J.P. Ortonne, et al . 1994. Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the gamma 2 subunit of nicein/kalinin (LAMININ-5). Nat. Genet. 6: 299-304 |
2. |
Bairoch, A., and
R. Apweiler.
1999.
The SWISS-Prot protein sequence data
bank and its supplement in 1999.
Nucleic Acids Res.
27:
49-54
|
3. | Bate, M. 1990. The mesoderm and its derivatives. In The Development of Drosophila melanogaster. M. Bate, and A. Martinez-Arias, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1013-1090. |
4. | Baumgartner, S., D. Martin, and R. Chiquet-Ehrismann. 1993. Drosophila ribosomal protein S19 sequence. Nucl. Acids Res 21: 3897 |
5. | Baumgartner, S., D. Martin, C. Hagios, and R. Chiquet-Ehrismann. 1994. ten-m, a Drosophila gene related to tenascin, is a new pair-rule gene. EMBO (Eur. Mol. Biol. Organ.) J. 13: 3728-3740 [Abstract]. |
6. | Bogaert, T., N. Brown, and M. Wilcox. 1987. The Drosophila PS2 antigen is an invertebrate integrin that, like the fibronectin receptor, becomes localized to muscle attachments. Cell. 51: 929-940 |
7. | Brabant, M.C., and D.L. Brower. 1993. PS2 integrin requirements in Drosophila embryos and wing morphogenesis. Dev. Biol 157: 49-59 |
8. | Brower, D.L., and S.M. Jaffe. 1989. Requirement for integrin during Drosophila wing development. Nature. 342: 285-287 |
9. |
Brower, D.L.,
T.A. Bunch,
L. Mukai,
T.E. Adamson,
M. Wehrli,
S. Lam,
E. Friedlander,
C.E. Roote, and
S. Zusman.
1995.
Non-equivalent requirements for PS1 and PS2 integrin at cell attachments in Drosophila; genetic
analysis of the ![]() |
10. |
Brown, N.H.,
D.L. King,
M. Wilcox, and
F.C. Kafatos.
1989.
Developmentally
regulated alternative splicing of Drosophila integrin PS2![]() |
11. |
Bunch, T.A., and
D.L. Brower.
1992.
Drosophila PS2 integrin mediates RGD-dependent cell-matrix interactions.
Development.
116:
239-247
|
12. | Burge, C., and S. Karlin. 1997. Prediction of complete gene structures in human genomic DNA. J. Mol. Biol 268: 78-94 |
13. | Burgeson, R., M. Chiquet, R. Deutzmann, P. Ekblom, J. Engel, H. Kleinman, G.R. Martin, G. Meneguzzi, M. Paulsson, J. Sanes, et al . 1994. A new nomenclature for the laminins. Matrix Biol. 14: 209-211 |
14. | Campos-Ortega, J.A., and V. Hartenstein. 1985. The Embryonic Development of Drosophila melanogaster. Springer-Verlag, Berlin. 9-84. |
15. |
Chi, H.-C., and
C.-F. Hui.
1989.
Primary structure of the Drosophila laminin B2
chain and comparison with human, mouse and Drosophila B1 and B2 chains.
J. Biol. Chem
264:
1543-1550
|
16. | Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem 162: 156-159 |
17. | Chung, A.E., R. Jaffe, J.P. Freeman, J.P. Vergnes, J.E. Braginsk, and B. Carlin. 1979. Properties of a basement membrane related glycoprotein synthesized by a mouse embryonal carcinoma-derived cell line. Cell. 16: 277-287 |
18. | Cserhalmi-Friedman, P.B., H. Baden, R.E. Burgeson, and A.M. Christiano. 1998. Molecular basis of non-lethal junctional epidermolysis bullosa: identification of a 38 basepair insertion and a splice site mutation in exon 14 of the LAMB3 gene. Exp. Dermatol 7: 105-111 |
19. | Drysdale, R., E. Rushton, and M. Bate. 1993. Genes required for embryonic muscle development in Drosophila melanogaster. Roux's Arch. Dev. Biol 202: 276-295 . |
20. | Ehrig, K., I. Leivo, W.S. Argaves, E. Ruoslahti, and E. Engvall. 1990. Merosin, a tissue-specific basement membrane protein, is a laminin-like protein. Proc. Natl. Acad. Sci. USA. 87: 3264-3268 [Abstract]. |
21. |
Fogerty, F.J.,
L.I. Fessler,
T.A. Bunch,
Y. Yaron,
C.G. Parker,
R.E. Nelson,
D.L. Brower,
D. Gullberg, and
J.H. Fessler.
1994.
Tiggrin, a novel Drosophila extracellular matrix protein that functions as a ligand for Drosophila
![]() ![]() |
22. |
Fristrom, D.,
P. Gotwals,
S. Eaton,
T.B. Kornberg,
M. Sturtevant,
E. Bier, and
J.W. Fristrom.
1994.
blistered: a gene required for vein/intervein formation
in wings of Drosophila.
Development.
120:
2661-2671
|
23. | Garcia-Alonso, L., R.D. Fetter, and C.S. Goodman. 1996. Genetic analysis of laminin A in Drosophila: extracellular matrix containing laminin A is required for ocellar axon pathfinding. Development 22: 2611-2621 . |
24. | Golic, K.G.. 1991. Site-specific recombination between homologous chromosomes in Drosophila. Science. 252: 958-961 |
25. | Gotwals, P.J., S.E. Paine-Saunders, K.A. Stark, and R.O. Hynes. 1994. Drosophila integrins and their ligands. Curr. Opin. Cell Biol. 6: 734-739 |
26. |
Graner, M.W.,
T.A. Bunch,
S. Baumgartner,
A. Kerschen, and
D.L. Brower.
1998.
Splice variants of the Drosophila PS2 integrins differentially interact
with the extracellular ligands Tiggrin, D-Laminin ![]() |
27. | Helbling-Leclerc, A., X. Zhang, H. Topaloglu, C. Cruaud, F. Tesson, J. Weissenbach, F.M. Tome, K. Schwartz, M. Fardeau, K. Tryggvason, et al . 1995. Mutations in the laminin alpha2-chain cause merosin-deficient congenital muscular dystrophy. Nat. Genet. 11: 216-218 |
28. |
Henchcliffe, C.,
L. Garcia-Alonso,
J. Tang, and
C.S. Goodman.
1993.
Genetic
analysis of laminin A reveals diverse functions during morphogenesis in
Drosophila.
Development.
118:
325-337
|
29. |
Horowitz, H., and
C.A. Berg.
1995.
Aberrant splicing and transcription termination caused by P element insertion into the intron of a Drosophila gene.
Genetics.
139:
327-335
|
30. | Hynes, R.O.. 1992. Integrins: versatility, modulation and signaling in cell adhesion. Cell. 69: 11-25 |
31. |
Karpen, G.H., and
A.C. Spradling.
1992.
Analysis of subtelomeric heterochromatin in the Drosophila minichromsome Dp1187 by single P element insertional mutagenesis.
Genetics.
132:
737-753
|
32. | Kiehart, D.P., and R. Feghali. 1986. Cytoplasmic myosin from Drosophila melanogaster. J. Cell Biol 103: 1517-1525 [Abstract]. |
33. | Kimmerly, W., K. Stultz, S. Lewis, K. Lewis, V. Lustre, R. Romero, J. Benke, D. Sun, G. Shirley, C. Martin, and M. Palazzolo. 1996. A P1-based physical map of the Drosophila euchromatic genome. Genome Res 6: 414-430 [Abstract]. |
34. | King, R.C. 1970. Ovarian development in Drosophila melanogaster. Academic Press Inc., New York. |
35. | Kivirikko, S., J.A. McGrath, C. Baudoin, D. Aberdam, S. Ciatti, M.G. Dunnill, J.R. McMillan, R.A. Eady, J.P. Ortonne, G. Meneguzzi, et al . 1995. A homozygous nonsense mutation in the alpha 3 chain gene of laminin 5 (LAMA3) in lethal (Herlitz) junctional epidermolysis bullosa. Hum. Mol. Genet 4: 959-962 [Abstract]. |
36. | Kusche-Gullberg, M., K. Garrison, A.J. MacKrell, L.I. Fessler, and J.H. Fessler. 1992. Laminin A chain: expression during Drosophila development and genomic sequence. EMBO (Eur. Mol. Biol. Organ.) J. 11: 4519-4527 [Abstract]. |
37. | Leptin, M., T. Bogaert, R. Lehmann, and M. Wilcox. 1989. The function of PS integrins during Drosophila embryogenesis. Cell. 56: 401-408 |
38. | Lindsley, D.L., and G. Zimm. 1992. The genome of Drosophila melanogaster. Academic Press Inc., London. p. 777. |
39. | Maniatis, T., E.F. Fritsch, and J. Sambrook. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. |
40. | Manning, G., and M.A. Krasnow. 1993. Development of the Drosophila tracheal system. In The Development of Drosophila melanogaster. M. Bate and A. Martinez-Arias, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 609-685. |
41. |
Miner, J.H.,
B.L. Patton,
S.I. Lentz,
D.J. Gilbert,
W.D. Snider,
N.A. Jenkins,
N.G. Copeland, and
J.R. Sanes.
1997.
The laminin ![]() ![]() ![]() |
42. |
Montagne, J.,
J. Groppe,
K. Guillemin,
M.A. Krasnow,
W.J. Gehring, and
M. Affolter.
1996.
The Drosophila serum response factor gene is required for
the formation of intervein tissue of the wing and is allelic to blistered.
Development.
122:
2589-2597
|
43. | Montell, D.J., and C.S. Goodman. 1988. Drosophila substrate adhesion molecule: sequence of laminin B1 chain reveals domains of homology with mouse. Cell. 53: 463-473 |
44. | Montell, D.J., and C.S. Goodman. 1989. Drosophila laminin: sequence of B2 subunit and expression of all three subunits during embryogenesis. J. Cell Biol 109: 2441-2453 [Abstract]. |
45. | Prokop, A., M.D. Martin-Bermudo, M. Bate, and N. Brown. 1998. Absence of PS integrins or laminin A affects extracellular adhesion, but not intracellular assembly, of hemiadherens and neuromuscular junctions in Drosophila embryos. Dev. Biol 196: 58-76 |
46. | Prout, M., Z. Damania, J. Soong, D. Fristrom, and J.W. Fristrom. 1997. Autosomal mutations affecting adhesion between wing surfaces in Drosophila melanogaster. Genetics. 46: 275-285 . |
47. | Pulkkinen, L., A.M. Christiano, T. Airenne, H. Haakana, K. Tryggvason, and J. Uitto. 1994. Mutations in the gamma 2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa. Nat. Genet 6: 293-297 |
48. | Rambukkana, A., J.L. Salzer, P.D. Yurchenco, and E.I. Tuomanen. 1997. Neural targeting of Mycobacterium leprae mediated by the G domain of the laminin-alpha2 chain. Cell. 88: 811-821 |
49. | Roote, C.E., and S. Zusman. 1995. Functions for PS integrins in tissue adhesion, migration and shape changes during early embryonic development in Drosophila. Dev. Biol 169: 322-336 |
50. |
Roote, C.E., and
S. Zusman.
1996.
Alternatively spliced forms of the Drosophila ![]() ![]() |
51. |
Samakovlis, C.,
N. Hacohen,
G. Manning,
D.C. Sutherland,
K. Guillemin, and
M.A. Krasnow.
1996.
Development of the Drosophila tracheal system occurs by a series of morphologically distinct, but genetically coupled branching events.
Development.
122:
1395-1407
|
52. | Sarras, M.P., L. Yan, A. Grens, X. Zhang, A. Agbas, J.K. Huff, P.L. St John, and D.R. Abrahamson. 1994. Cloning and biological function of laminin in Hydra vulgaris. Dev. Biol 164: 312-324 |
53. |
Stark, K.A.,
G.H. Yee,
C.E. Roote,
E.L. Williams,
S. Zusman, and
R.O. Hynes.
1997.
A novel alpha integrin subunit associates with ![]() |
54. | Tautz, D., and C. Pfeiffle. 1989. A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals a translational control of the segmentation gene hunchback. Chromosoma. 98: 81-85 |
55. | Timpl, R., and J.C. Brown. 1996. Supramolecular assembly of basement membranes. BioEssays. 18: 123-132 |
56. | Timpl, R., H. Rohde, P.G. Robey, S.I. Rennard, J.M. Foidart, and G.R. Martin. 1979. Laminin-a glycoprotein from basement membranes. J. Biol. Chem 254: 993-997 . |
57. | von Heijne, G. 1986. A new method for predicting signal sequence cleavage sites. Nucl. Acids Res. 14:4683-4690. |
58. |
Walsh, E.P., and
N.H. Brown.
1998.
A screen to identify Drosophila genes required for integrin-mediated adhesion.
Genetics.
150:
791-805
|
59. | Wieschaus, E., C. Nüsslein-Volhard, and G. Jürgens. 1984. Mutations affecting the pattern of the larval cuticle in D. melanogaster. III. Zygotic loci on the X chromosome. Roux's Arch. Dev. Biol 193: 296-307 . |
60. | Wieschaus, E., and C. Nüsslein-Volhard. 1986. Looking at embryos. In Drosophila: a Practical Approach. D.B. Roberts, editor. IRL Press, Washington D.C. pp. 199-227. |
61. |
Woodruff, R., and
M. Ashburner.
1979.
The genetics of a small autosomal region of Drosophila melanogaster containing the structural gene for alcohol
dehydrogenase. II. Lethal mutations in the region.
Genetics.
92:
133-149
|
62. |
Xu, H.,
X.-R. Wu,
U.M. Wewer, and
E. Engvall.
1994.
Murine muscular dystrophy caused by a mutation in laminin ![]() |
63. | Yarnitzky, T., and T. Volk. 1995. Laminin is required for heart, somatic muscles and gut development in the Drosophila embryo. Dev. Biol. 169: 609-618 |
64. | Young, P.E., and D.P. Kiehart. 1991. Nonmuscle myosin is required throughout Drosophila development. J. Cell Biol 115: 184-193 . |
65. | Yurchenco, P.D., and Y.S. Cheng. 1993. Self-assembly and calcium-binding sites in laminin. A three-arm interaction model. J. Biol. Chem. 26: 7286-7299 . |
66. |
Yurchenco, P.D.,
Y. Quan,
H. Colognato,
T. Mathus,
D. Harrison,
Y. Yamada, and
J.J. O'Rear.
1997.
The alpha chain of laminin-1 is independently secreted and drives secretion of its beta- and gamma-chain partners.
Proc.
Natl. Acad. Sci. USA.
94:
10189-10194
|
67. |
Zhang, X.,
R. Vuolteenaho, and
K. Tryggvason.
1996.
Structure of the human
laminin ![]() |
68. | Zusman, S., K.R. Patel, C. Ffrench-Constant, and R.O. Hynes. 1990. Requirements for integrins during Drosophila development. Development. 108: 391-402 [Abstract]. |