1 Program in Cell Structure and Development, Medical Biotechnology Center,
University of Maryland Biotechnology Institute, 725 West Lombard Street,
Baltimore, MD 21201, USA
2 Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120
Heidelberg, Germany
* Author for correspondence (e-mail: vogel{at}umbi.umd.edu)
Accepted 20 July 2005
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SUMMARY |
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Key words: Extracellular matrix, Basement membrane, Hemidesmosome, C. elegans, Cell adhesion
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Introduction |
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The structure of vertebrate fibulin-1 gene products has revealed the
presence of conserved alternate splice forms, fibulin-1C and fibulin-1D;
however, little is known about the specific functions that would justify their
conservation throughout metazoan evolution. Specific roles have been suggested
for fibulin-1C in tumor progression (Moll
et al., 2002) and for fibulin-1D in tumor suppression,
synpolydactyly and giant platelet syndrome
(Qing et al., 1997
;
Debeer et al., 2002
;
Toren et al., 2003
). These
observations, in addition to data showing that fibulin-1C binds to the
basement membrane glycoprotein nidogen at 30-fold higher affinity than does
fibulin-1D, suggest that these two splice variants have distinct functions
(Sasaki et al., 1995
).
C. elegans has a single fibulin gene that is a highly conserved
ortholog of vertebrate fibulin-1. Mutations in C. elegans fibulin-1
result in gonad morphogenesis defects, but can also suppress gonad
morphogenesis defects associated with GON-1 and MIG-17 ADAM metalloproteases
(Hesselson et al., 2004;
Kubota et al., 2004
). One
model based on these studies suggests that fibulin-1 and GON-1 act in
opposition to one another in the respective inhibition or promotion of tissue
expansion (Hesselson et al.,
2004
).
Structural conservation of the nematode fibulin-1 gene extends to the
presence of exons encoding the alternate isoforms fibulin-1C and fibulin-1D
(Barth et al., 1998). To
investigate the functions of individual fibulin splice variants, we have
determined the structure, localization and loss-of-function phenotype specific
to each. We present data indicating that these two splice variants have
distinct roles during C. elegans morphogenesis. Fibulin-1C regulates
cell shape and adhesion within developing pharynx, gonad, intestine and muscle
tissues, whereas fibulin-1D assembles in flexible polymers that connect the
pharynx and body-wall-muscle basement membranes. In addition, both variants
are dependent on another extracellular matrix (ECM) protein, hemicentin, for
assembly at hemidesmosome-mediated, mechanosensory neuron and uterine
attachments to the epidermis. In C. elegans, hemicentin assembles
into line-shaped structures that are adhesive and flexible
(Vogel and Hedgecock, 2001
).
Although little is known about the distribution or function of the two
vertebrate hemicentin orthologs, a mutation in human hemicentin-1 was recently
implicated in age-related macular degenerative disease
(Schultz et. al., 2003
).
We suggest that the association between these two conserved ECM proteins is likely to be conserved in non-nematode species, and that fibulin splice variants have distinct but complementary roles in tissue assembly and organization that are likely to have been conserved in other species as well.
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Materials and methods |
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Isolation and characterization of full-length fibulin-1C and fibulin-1D cDNAs
After reverse transcription of RNA isolated from mixed stages, cDNA from
fbl-1C and fbl-1D was cloned using primers specific for the
5' and 3' ends of the predicted coding regions. After sequencing
full-length clones, forward primers specific for exon 5C
(5'-ATTGATGAGTGTGCCACACTG-3') or exon 5D
(5'-CGTAACGAATGTTTAACCCGC-3') were used in combination with
reverse primers specific for exon 14 (5'-CTATCGAATCTTCATGAGCGG-3')
and exon 16 (5'-TTAAAATGGATACTTTGAAAC-3') to amplify reverse
transcribed cDNA in order to confirm the structure of sequenced transcripts
and to detect the presence of other splice variants.
Constructs and plasmids
PCR fragments were generated with Pfu Turbo DNA polymerase
(Stratagene), using cosmid DNA as templates. Cosmids F56H11 and T05A1 were
used to amplify all fibulin constructs. The fbl-1 rescuing construct
was amplified from nucleotide 13,748 in F56H11 to nucleotide 3154 in T05A1.
Fibulin promoter GFP constructs used 5' regulatory sequences
F56H11:13,748-15,683. Y47D3B was used to amplify the sbp-1 regulatory
region from nucleotide 16,731 to nucleotide 18,775. Y73C8B was used to amplify
the lag-2 promoter region, from nucleotide 6,395 to nucleotide 8,415.
The unc-54 promoter was obtained from vector pPD30.38. GFP and YFP
coding sequences were obtained from vectors pPD113.37 and pPD132.112,
respectively. GFP was inserted, in separate constructs, after nucleotide
F56H11:15,892 in exon 2, after nucleotide T05A1:1,039 in exon 14, and after
nucleotide T05A1:2,023 in exon 16. Stop codons and reading-frame shifts were
introduced after nucleotide F56H11:17,565 to inactivate fbl-1C, or
after nucleotide F56H11:16,872 to inactivate fbl-1D. All constructs
were cloned in pGEM vector plasmid (Promega).
Transgenic rescue and expression studies
Transgenic lines were made by microinjection. All constructs were injected
at a concentration of 40 ng/µl, together with pRF4 (a plasmid containing a
rol-6 dominant mutation) at 100 ng/µl.
Confocal, DIC and fluorescence microscopy
DIC and fluorescence images were obtained with an Olympus BX51 light
microscope and a Magnafire camera. Confocal images were obtained with a Zeiss
LSM 510 META microscope.
Electron microscopy
Adult hermaphrodites were fixed and embedded for serial thin-section
electron microscopy according to Hall
(Hall, 1995).
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Results |
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Fibulin alleles, loss-of-function phenotype
A deletion mutant, fbl-1(hd43), was identified in a PCR-based
screen of a C. elegans mutant library (see Materials and methods).
The deleted region includes nucleotides F56H11:15,472 to 16,582, removing
sequences that encode the initiator methionine, the signal sequence, three
anaphylatoxin modules and the first EGF, in addition to 209 base pairs of
5' sequence upstream of the initiator methionine codon
(Fig. 1A). The mutant animals
are likely to be molecular nulls, sharing several penetrant phenotypic defects
in cell adhesion and morphology that are rescued completely in mutant animals
containing a fibulin transgene that includes nucleotides F56H11:13,748 to
T05A1:3,154 (see below and Table
1).
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In wild-type C. elegans male gonads, the distal tip cells are stationary and the migratory leader cell found at the proximal end of the gonad is the linker cell. In fbl-1(hd43) mutant males, linker cell migration appears to be unaffected, and gonad morphogenesis proceeds normally from early L2 larval stage through early L4. However, in late L4 males, the seminal vesicle ruptures and separates from the vas deferens, severing the pathway between testis and cloaca (Fig. 2C,D). As a result, fbl-1(hd43) adult males are sterile.
Pharynx
DIC examination of the pharynx of fbl-1 mutants reveals that it is
asymmetrical and may not function properly for feeding because animals are
also small (about 60% of the wild-type body length) and grow slowly.
Pharyngeal defects appear to be relatively mild in earlier larval stages, but
increase in severity in adults, with the pharynx appearing increasingly
asymmetric and also appearing wider and shorter than in wild type
(Fig. 2E,F). Electron
microscopy reveals that the shape of the basal surface of pharyngeal cells is
deformed, particularly around pharyngeal muscle cells
(Fig. 3C,D). It is likely that
the defects originate during pharyngeal morphogenesis or that they result from
mechanical tension caused by a contraction of pharyngeal muscle that distorts
a weakened pharyngeal basement membrane.
Body-wall muscle
Although hd43 animals are lethargic, they are able to make
coordinated movements when prodded lightly. Examination of body-wall muscle
attachments with a functional GFP-integrin ß pat-3 reporter
(Plenefisch et al., 2000)
reveals that integrin-based attachments of body-wall muscle to the epidermis,
including dense body organization and muscle cell polarity, which are
defective in other ECM mutants (Rogalski
et al., 1993
; Huang et al.,
2003
), appear for the most part normal. However, gaps are found at
nearly all (>95%) muscle-muscle junctions, and mutant cells have a compact,
rhomboid shape in contrast to the elongated, spindle appearance found with
wild-type muscle (Fig.
2G,H).
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Uterine attachment
A complex junction forms where a multinucleate uterine cell, utse, attaches
to epidermal seam cells. In fbl-1 mutants, the assembly of the
attachment at this site is defective as viewed by a defect in hemicentin
assembly (Fig. 10I,J). In
contrast to him-4 mutants, where the uterus prolapses during
egg-laying, few fertile eggs enter the uterus in fbl-1(hd43) animals.
Because there is little or no egg-laying in most fbl-1 animals, the
mechanical stress on the uterus is reduced and uterine prolapse is rare.
Fibulin expression by intestine, muscle and hypodermal cells
A transcriptional fusion with the fibulin-1 5' regulatory sequence
(F56H11:13,748-15,683) driving expression of a GFP reporter was used to
determine sites of fibulin synthesis. The same 5' regulatory sequence
was used in the fibulin constructs that rescue all of the phenotypes described
below (Table 1). Expression was
most dramatic in the anterior and posterior two to four intestinal cells, head
and tail body-wall muscles, and in the hypodermal syncytium
(Fig. 4). Intestinal expression
is detected in late embryogenesis and gradually decreases in anterior
intestinal cells until it is not detectable in adults, but it persists in the
posterior intestinal cells well into adulthood. Hypodermal, and head and tail
body-wall muscle, expression is also detected late in embryogenesis and
persists into adulthood. In the hermaphrodite gonad, expression is seen in the
spermatheca, starting in young adult hermaphrodites and remaining strong in
older adults. Rare expression is also detected in three to five unidentified
neurons in the nerve ring.
Fibulin splice forms have distinct localizations and functions
A transgene comprising the same 5' regulatory sequence that was used
to determine the sites of synthesis (Fig.
4) driving expression of a full-length fibulin gene can rescue
each of the phenotypic defects described above
(Table 1). A GFP or YFP tag was
added to each fibulin-1 splice form at either the N or C terminal (see
Materials and methods for a description of constructs). The splice
form-specific constructs were expressed in fbl-1(hd43) mutant animals
to determine localization and the ability of each splice form to rescue each
mutant phenotype. Fibulin-1C with a GFP tag at either end localizes to male
and hermaphrodite gonad, anterior and posterior intestine and pharyngeal
basement membranes, body-wall muscle, GLR cells, uterine attachment and
mechanosensory neurons (Fig.
5A-H). On body-wall muscle, fibulin-1C accumulates at dense
bodies, but is most intense between muscle cells, particularly between
anterior and posterior pairs of muscle cells. Fibulin-1C is detectable on the
surface of each of these cell types from late embryonic stages onwards, with
the exception of uterine attachments, where fibulin-1C::GFP becomes detectable
as the uterine attachment develops in late L4. Fibulin-1C::GFP is able to
completely rescue the size, pharynx, uterine and muscle attachment, and gonad
migration phenotypes, and to partially rescue mechanosensory neuron attachment
defects (see Table 1).
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Because we were not able to detect distal tip cells as a source of fibulin synthesis, this raised the possibility that fibulin might not be expressed by the migrating gonadal leader cell, but by its migration substratum. We expressed fibulin under the control of promoters specific for the muscle substratum (unc-54), intestine (sbp-1) and distal tip cells (lag-2) and found that only intestinal expression of fibulin completely rescues the gonad morphology defect in addition to defects in intestine, pharynx and body-wall muscle; it also partially rescues mechanosensory neuron and uterine attachment defects (Table 1). Fibulin-1::GFP secreted from intestinal cells is detected on each of these tissues.
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To determine whether the assembly of either of the fibulin-1 splice
variants found in these structures are dependent on hemicentin, we examined
fibulin localization in the presence and absence of hemicentin. In a
hemicentin null [him-4(rh319)] background, fibulin-1C and fibulin-1D
are nearly undetectable at mechanosensory neuron and uterine attachments
(Fig. 8B,D,
Fig. 9B,E), and fibulin-1D is
undetectable in flexible tracks connecting pharyngeal and body-wall-muscle
basement membranes (Fig. 8F).
We also examined the assembly of fibulin-1 variants in the absence of another
extracellular protein known to be required for mechanosensory neuron
attachments, MEC-1 (Emtage et al.,
2004). In mec-1(e1066) animals, fibulin-1 distribution on
mechanosensory neurons is not detectably different from fibulin distribution
in wild-type animals (cf. Fig.
9A,C with
9D,F).
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Discussion |
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Fibulin-1C appears to have specific roles in regulating the shape and
adhesion of cells in the developing pharynx, intestine, body-wall muscle and
gonadal tissue. Recent studies show that mutations in fibulin-1 suppress the
gonad migration phenotypes seen in metalloprotease defective gon-1
and mig-17 mutants. They also suggest a functional interaction
between fibulin-1 and ADAM metalloproteases
(Hesselson et al., 2004;
Kubota et al., 2004
), and are
consistent with a specific role for fibulin-1C in regulating the shape of the
hermaphrodite gonad. Fibulin-1C is also necessary for the assembly of
hemicentin, which is required for hemidesmosome-mediated uterine attachments.
Although fibulin-1D is also found in this location, it can only partially
compensate for the lack of fibulin-1C.
By contrast, fibulin-1D appears to have a single unique role in the assembly of the flexible, hemicentin-containing tracks found joining the pharynx and body-wall-muscle basement membranes. Although fibulin-1C is also found in the flexible tracks, fibulin-1D is necessary and sufficient for these structures; loss of fibulin-1C has no effect on these structures and cannot compensate for the lack of fibulin-1D in this location. Complete rescue of the mechanosensory neuron attachment defects in fbl-1(hd43) animals requires the presence of both fibulin-1C and fibulin-1D, suggesting that at some hemidesmosome-mediated attachment sites these two splice forms can function in tandem. Because both are required, it seems that their functions are distinct in this location as well. An alternate possibility is that the effect is a function of dose: either splice form may rescue alone if it is present in sufficient quantity. However, because we are already expressing each splice form at high levels in transgenic animals, this possibility seems less likely.
The source of fibulin for many tissues appears to be at or near the site of assembly. It was therefore unexpected to find that fibulin-1 expressed ectopically under the control of a distal tip cell promoter did not rescue gonad morphogenesis or any other defects, and that expression of fibulin-1 under control of a muscle-specific promoter rescued assembly of flexible hemicentin tracks but did not rescue the muscle attachment (or any other) defects. Instead, we found that the fibulin-1 gene was able to rescue gonad migration and most other defects when expressed under the control of an intestine-specific promoter (Table 1). We observed that intestinally expressed fibulin::GFP was secreted and accumulated on the surface of intestine, gonad, pharynx and body-wall muscle cells. This pattern is identical to the pattern seen for fibulin-1C under control of its endogenous promoter. This suggests that fibulin-1C must assemble into a complex or be modified in some other way by intestinal cells for proper secretion, localization and/or function.
Fibulin-1 assembly is hemicentin dependent in some locations
In vertebrates, fibulin-1 (and other fibulins) interacts directly with a
large number of ECM proteins, including nidogen and laminin
(Argraves et al., 2003;
Timpl et al., 2003
). Although
it is not known whether the interactions between fibulin-1 and hemicentin
described here are direct or indirect, fibulin-1C and fibulin-1D splice
variants require hemicentin for localization and assembly in multiple tissue
locations, including flexible tracks, and mechanosensory neuron and uterine
attachments. Conversely, hemicentin localization to mechanosensory neurons and
uterine attachments is independent of fibulin-1. However, hemicentin assembled
at these sites has a discontinuous and frayed appearance in the absence of
both fibulin-1 isoforms. Therefore, one function of fibulin-1 at these sites
appears to be to refine the hemicentin localized there into smooth continuous
tracks. The lack of a complete overlap in the localization of hemicentin and
fibulin in these locations suggests that there may be other proteins involved
in the assembly of these structures, but it does not exclude the possibility
that fibulins and hemicentin can interact directly. In locations other than
uterine and mechanosensory neuron attachments and flexible tracks, hemicentin
and fibulin-1 variants can assemble and function independently of one
another.
MEC-1, a secreted protein composed of EGF and Kunitz repeats is an obvious
candidate to interact with fibulin at mechanosensory neuron attachments
(Emtage et al., 2004). Like
hemicentin, MEC-1 is required for the assembly of mantle, hemidesmosomes and
associated intermediate filaments at mechanosensory neuron attachments to the
epidermis. Although we found that fibulin-1C and fibulin-1D assembly at these
attachments is hemicentin dependent, loss of MEC-1 from these structures has
no detectable effect on fibulin assembly. This indicates that fibulin assembly
is dependent on the presence of hemicentin, but is not dependent on the
presence of hemidesmosome-mediated attachments, mantle, MEC-1 or the large
number of other proteins likely to be found in these structures. Conversely,
MEC-1 is required for the assembly and function of mechanosensory channel
complexes, whereas fibulin and hemicentin appear to have no direct role in the
assembly and function of these complexes or in mechanotransduction,
reinforcing earlier observations that mechanosensory neuron anchorages are not
required for mechanotransduction (Vogel
and Hedgecock, 2001
; Emtage et
al., 2004
).
Relevance to vertebrate fibulins
Fibulin-1 is associated with basement membranes and elastic fibers in
vertebrates. The fibulin-1 phenotypes seen in the blood vessels of mice where
the fibulin-1 gene has been inactivated, including the dilated and irregular
lumen and the disruption in endothelial morphology and cell-cell-contacts
(Kostka et al., 2001), are
reminiscent of the fibulin-1C specific defects in gonad, pharynx, intestine
and muscle cell adhesion and morphology seen here. Based on this striking
similarity, a reasonable speculation is that the blood vessel defects observed
in the fibulin-1-deficient mouse are primarily due to loss of the fibulin-1C
splice variant. It is tempting to speculate further that similar to C.
elegans fibulin-1C, vertebrate fibulin-1C is preferentially distributed
in basement membranes and fibulin-1D is preferentially distributed in elastic
fibers. This model is consistent with data showing that fibulin-1C binds to
the basement membrane glycoprotein nidogen with a 30-fold higher affinity than
does fibulin-1D (Sasaki et al.,
1995
). However, specific protein localization data for vertebrate
fibulin-1 splice variants have not yet been determined and will require
variant specific reagents to discriminate between the fibulin-1 isoforms.
Nematode fibulin-1C and fibulin-1D variants differ not only in their
C-terminal sequences, but also have a single alternately spliced EGF repeat.
We do not know whether the functional differences between the two splice
variants are a result of the different EGF, C-terminal modules, or a
combination of both. Although alternate splicing of vertebrate fibulin-1D EGF
repeats has not been reported, vertebrate fibulins 2 and 3 have single EGF
repeats that are alternately spliced in a tissue-specific manner
(Lecka-Czernik et al., 1995;
Grassel et al., 1999
). It is
possible that after duplication of the ancestral, founding fibulin gene in the
chordate lineage, alternately spliced exons encoding EGF repeats were lost in
fibulin-1 but retained in fibulins 2 and 3, or that alternately spliced
fibulin-1 transcripts exist that have not yet been reported in the literature.
Either way, it is likely that the functional consequences of alternate EGF
splicing observed in nematodes extends to vertebrate fibulins
(Lecka-Czernik et al., 1995
;
Grassel et al., 1999
).
Hemicentin and fibulin-1 are the only two proteins that have been localized to flexible tracks connecting the pharynx and body-wall-muscle basement membranes. The flexible tracks pivot, flex and stretch as the worm forages. It is possible that these tracks are unique structures specific to nematodes, a precursor to the elastic fibers that evolved further in the vertebrate lineage, or that they represent new structures yet to be described in vertebrates.
Fibulins and hemicentins share a region of high homology at the C terminus
of both proteins that contains EGF and FC modules. Based on this homology,
human hemicentin-1 has occasionally been included as an atypical member of the
fibulin family and referred to as fibulin-6. In C. elegans, this
region in the hemicentin protein appears to be involved in hemicentin track
assembly (C.D. and B.E.V., unpublished) and may provide a potential mechanism
for the interaction between fibulin and hemicentin. We speculate that the
hemicentin-dependent assembly of fibulin-1 variants observed in some nematode
tissues may have a broader relevance for hemicentin and fibulin orthologs in
vertebrates. Although a relationship between the vertebrate orthologs of these
genes has not been demonstrated, a potential link is suggested by the
observation that human hemicentin-1, fibulin-3 and fibulin-5 have recently
been implicated in multiple forms of human macular dystrophy
(Stone et al., 1999;
Stone et al., 2004
;
Schultz et al., 2003
).
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ACKNOWLEDGMENTS |
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