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First published online December 8, 2005
doi: 10.1242/10.1242/dev.02177
1 Department of Genetics, Max-Planck-Institute for Developmental Biology,
Tübingen, Germany
2 Department of Medical Biochemistry, Gothenburg University, Gothenburg,
Sweden.
Authors for correspondence (e-mail:
anne.uv{at}medkem.gu.se;
bernard.moussian{at}tuebingen.mpg.de)
Accepted 25 October 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Tubulogenesis, Trachea, Epidermis, Cuticle, Chitin, Drosophila, Apical ECM, Knk, Septate junction
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Spicer and Tien, 2004;
Toole, 2004), and cellulose
fibre orientation in plants controls the directionality of cell expansion
(Roudier et al., 2005). In
addition, an essential role for chitin in epithelial tube size regulation in
the developing fly trachea was recently discovered
(Tonning et al., 2005). The
tracheal system stems from epithelial sacks that invaginate from the epidermis
and ramify to generate tubes of distinct dimensions
(Ghabrial et al., 2003;
Uv et al., 2003). Like many
developing tubes, the newly formed tracheal branches are narrow, and
subsequent tube expansion to attain functional lumen sizes requires
coordinated cell rearrangements and apical membrane growth
(Beitel and Krasnow, 2000).
Tracheal chitin is synthesized and deposited into the lumen by Chitin synthase
1 (CS-1), and loss of tracheal chitin causes severe lumen diameter
irregularities, with local dilations and cysts. Luminal chitin assembles into
a defined transient cable that expands in unison with lumen diameter growth,
suggesting that the shape and organization of the chitin matrix is crucial for
uniform tube growth (Tonning et al.,
2005).
Chitin is a long linear sugar formed by transmembrane enzymes that link
cytosolic UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) into chains of repeating
GlcNAc residues that extrude from the apical cell surface
(Cohen, 2001). CS-1 is encoded
by krotzkopf verkehrt (kkv) and is responsible for chitin
deposition also in the embryonic cuticle
(Ostrowski et al., 2002;
Moussian et al., 2005a).
Within the stratified cuticle, chitin is found as a lamellar structure in the
layer abutting the epidermal cells, called the procuticle. These lamellae are
built from sheets of chitin microfibrils and are tightly packed to confer
stability and elasticity to the exoskeleton, as well as assisting in the
concurrent deposition of the overlaying protein-rich epicuticle
(Merzendorfer, 2005;
Moussian et al., 2005a). The
architecture of the tracheal luminal chitin matrix is not known, but here
chitin also appears organized into long filaments that run parallel with tube
length (Tonning et al., 2005).
Many previously identified tracheal tube expansion mutants exhibit an abnormal
intraluminal chitin fibre, where matrix diameter and texture are affected
(Tonning et al., 2005). These
mutants disrupt genes that encode septate junction (SJ) proteins
(Behr et al., 2003;
Paul et al., 2003;
Llimargas et al., 2004;
Wu et al., 2004) and develop
overgrown tracheal lumens. SJs share some functions and components with
vertebrate tight junctions, but localize to the lateral cell surface, and
their requirements for luminal chitin fibre organization and tube expansion
are unclear (Wu and Beitel,
2004).
In order to understand more about chitin matrix assembly and regulated
tracheal tube expansion, we tested whether mutations known to disrupt cuticle
differentiation could affect the tracheal luminal chitin matrix. Here we show
that the two mutants knickkopf (knk) and
retroactive (rtv), which display cuticle phenotypes that
parallel that of krotzkopf verkehrt (kkv)
(Jurgens et al., 1984;
Wieschaus et al., 1984;
Ostrowski et al., 2002;
Moussian et al., 2005a),
develop severe tracheal tube size defects that are reminiscent of those of
chitin-deficient embryos. Rtv encodes a transmembrane protein with putative
chitin-binding properties and is required for lamellar procuticle organization
(Moussian et al., 2005b). We
find that Knk and Rtv are specifically required for chitin filament assembly
in the tracheal lumen and cuticle. Further analysis of Knk uncovers an
essential role for the tracheal epithelium in organizing extracellular
chitinous matrices.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
unless otherwise noted. The following primary antibodies were used: tracheal
lumen specific antibody mouse monoclonal IgM 2A12 [1:10; Developmental Studies
Hybridoma Bank (DSHB)], rabbit anti-ß-gal (1:500; Jackson), rabbit
anti-GFP (1:500; Molecular Probes), mouse IgG1 monoclonal anti-Crumbs (1:10;
DSHB), rabbit IgG anti-Pio (1:20; provided by M. Affolter), mouse IgG
monoclonal anti-Discs large 1 (1:10; DSHB) and mouse monoclonal IgG2a
anti-Fasciclin 3 (1:10;DSHB). The Knk antiserum was generated by immunizing
rabbits with a fusion protein containing amino acids 33 to 484 of Knk fused to
a His-tagged polypeptide (Qiagen). The anti-Knk antiserum was pre-absorbed
against wild type embryos (stages 0-13) before use (1:1500). For fluorescent
visualization the following secondary antibodies from Molecular Probes were
used at 1:500 dilution: Alexa 488 goat anti-rabbit IgG, Alexa 488 goat
anti-mouse IgM, Alexa 594 goat anti-mouse IgM, Alexa 546 goat anti-mouse IgG,
Alexa 546 goat anti-rabbit IgG, Alexa 555 goat anti-mouse IgG and Alexa 555
goat anti-rabbit IgG. For labelling of tracheal luminal chitin, embryos were incubated with a FITC-conjugated chitin-binding probe (CBP; New England BioLabs) at 1:500 dilution for 3 hours together with the secondary antibody, and mounted in Prolong anti-fade (Molecular Probes). A BIO RAD RADIENCE 2000 was used to obtain confocal images, and Nikon Eclipse E1000 was used for obtaining fluorescent images. Images were processed in Adobe Photoshop 7.0.
Fly strains and chemical treatments
The mutant alleles of kkv, knk and rtv used in this study
were kkv1, knk7A69, knk5C77,
rtv11 and rtvBNd, which are amorphic ethyl
methanesulfonate (EMS)-induced mutations
(Jurgens et al., 1984;
Wieschaus et al., 1984;
Ostrowski et al., 2002;
Moussian et al., 2005b). The
other tube expansion mutants used were megaG0012
(pckG0012 - FlyBase; obtained from Bloomington Stock
Collection), fas2EB112
(Grenningloh et al., 1991),
grhs2140 (Spradling,
1999) and grhIM. The UAS-lines
UAS-knk and UAS-knkTM were generated by standard
germline transformation. UAS-knk contains the knk open
reading frame obtained by RT-PCR from pupal extract. UAS-knkTM
contains a sequence encoding the N-terminal 667 amino acids of Knk (i.e.
omitting the last predicted recognition domains required for GPI-modification)
fused to a sequence encoding the C-terminal 17 amino acids transmembrane
domain of Transferrin (CG10620). Sequences of CG10620 that could code for the
predicted recognition domains required for GPI-modification were not included.
The two GAL4-lines used were Btl-Gal4 line
(Shiga et al., 1996), which
expresses GAL4 in all tracheal cells from stage 12-13, and Tub-GAL4,
which expresses ubiquitous GAL4. In all experiments CyO, TM3 and
FM7c balancer strains carrying GFP or lacZ
transgenes were used as necessary to identify embryos with the desired
genotypes.
Inhibition of chitin synthesis was achieved with Nikkomycin Z (Sigma-Aldrich). Nikkomycin was diluted in water to generate a stock solution of 10 mg/ml, mixed with heat-inactivated yeast paste (1:10) to generate a final concentration of 1 mg/ml, and placed on a piece of parafilm on apple plates on which the flies could feed. Flies were fed Nikkomycin-containing yeast for 2 days before embryo collections.
In-situ hybridization
Whole-mount in-situ hybridizations were performed with digoxigenin-labelled
RNA sense and antisense probes as described
(Tonning et al., 2005). RNA
probes for knk were generated using the knk cDNA RE24065 as
template, and were hydrolyzed to yield RNA probes of approximately 500
bases.
Histology
Fully developed embryos were dechorionated manually, devitellinized by
shaking in 100% methanol, and incubated over night at 65°C in Hoyer's
medium mixed with lactic acid (1:1)
(Wieschaus and Nüsslein-Volhard,
1986). Embryos were analysed by fluorescence and light microscopy
using a Zeiss Axiophot. Transmission electron microscopy (TEM) analysis was
performed as described by Moussian and colleagues
(Moussian et al., 2005a). For
gold labelling of chitin, we used biotinylated Wheat Germ Agglutinin (WGA;
1:500, Vector Laboratories), which was recognized by an anti-biotin antibody
(1:300, Enzo Diagnostics), which in turn was detected by protein A conjugated
to 10-nm gold particles (1:100). Specimens labelled with gold were contrasted
for only 3 minutes instead of 10 minutes.
Molecular biology
For protein sequence analysis and comparisons, tools and software at the
Expasy proteomics server were used
(http://www.expasy.org).
The mutant alleles of knk were sequenced using PCR fragments
amplified from homozygous knk genomic DNA as templates. For each
allele at least two independent PCR-reactions were sequenced.
Western blot protein analysis
Larval membrane preparations were prepared with the Pierce Mem-PER
Eukaryotic Membrane Protein Extraction Kit using protocol 3 for soft tissue,
and detergents were removed using the PAGEprep Advance Kit from Pierce. For
western blot experiments, the Knk antiserum was used in a dilution of 1:2000
on 15 µg of embryo extracts, rabbit anti-Tout-velu (Ttv) at 1:1500 and anti
Syx1A (DSHB) at 1:5. For detection, we used Western lightning (Perkin Elmer)
with a donkey-HRP-conjugated secondary antibody (1:5000, Amersham). Digestion
with Phospholipase C from Bacillus cereus (Sigma) was performed on
membrane extracts from stage 17 embryos and as proposed by the enzyme
supplier.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wieschaus et al., 1984;
Ostrowski et al., 2002;
Moussian et al., 2005a;
Moussian et al., 2005b). We
therefore asked if knk and rtv might have additional roles
in tracheal tube size regulation. Two predicted knk null-alleles
(knk7A69 and knk5C77) and one
rtv null allele (rtv11) were analysed for
tracheal tube shapes using the tracheal lumen specific antibody 2A12. Indeed,
both knk and rtv mutant embryos develop anomalous tube
dimensions without affecting the branch pattern of the tracheal network
(Fig. 1A-C).
The 2A12 antigen is expressed by the tracheal epithelium and secreted into
the lumen from the onset of tube diameter growth
(Beitel and Krasnow, 2000), and
in the major dorsal trunks (DT) (where two to six cells surround the lumen
circumference; Fig. 1P) the
2A12 antigen is expressed from embryonic stage 14
(Fig. 1D). At this stage the
developing trachea in both knk and rtv mutants display weak
and diffuse luminal 2A12 levels, although the typical cellular levels of this
expansion marker appear normal (Fig.
1D-F). During the following 3 hours (stage 15), the wild-type DT
diameter expands 3- to 5-fold in a highly regulated manner to form uniform
tubes (Fig. 1G,P). In both
knk and rtv mutants this tube expansion is impaired; the DT
fusion branch lumens, which are formed by specialized toroidal cells
(Fig. 1P), do not expand as
much as in the wild type (arrows in Fig.
1G-I), and the remaining DT lumen dilate excessively in both
mutants to form tubes with a cystic appearance. The degree of DT diameter
defects is weaker in rtv than in knk mutants (compare also
Fig. 2B,C). After diameter
growth at stage 15, the DTs of both mutants also become excessively elongated
and convoluted (Fig. 1J-L). In
addition, the narrow and extended ganglionic branches (GB) display
discontinuous 2A12 labelling at the entry-point into the ventral nerve cord
(arrowheads in Fig. 1M-O,Q),
and local lumen dilations are also seen in narrower multicellular tracheal
tubes (not shown). Thus, knk and rtv are two tube size
mutants required for (1) uniform tube expansion, (2) restricted tube
elongation and (3) lumen integrity in a subset of tracheal epithelial tubes.
As lumen dimension relates to the size of the apical epithelial surface, the
disrupted tube diameter and lengths in knk and rtv mutants
probably reflects uncoordinated apical cell surface expansion within their
tracheal epithelium.
|
), whereas the DT fusion branch constrictions and tube
dilations are less severe in rtv mutants
(Fig. 2B-D). If Knk and Rtv
function in a chitin-dependent pathway, the simultaneous disruption of CS-1
activity and either Knk or Rtv would not be predicted to cause enhanced
tracheal defects, compared with embryos only lacking chitin. We used the
specific chitin synthase inhibitor Nikkomycin, a GlcNAc substrate analogue
that acts as a competitive inhibitor of chitin synthases
(Cabib, 1991;
Tellam et al., 2000) to
inhibit chitin synthesis in knk and rtv mutants. Wild-type
flies fed with a Nikkomycin dose of 1 mg/ml produce embryos that reproduce all
aspects of the kkv mutant phenotype
(Fig. 2A,D and not shown). We
find that knk and rtv mutant embryos produced by flies
treated with the same Nikkomycin dose develop tracheal defects
indistinguishable from those of Nikkomycin-treated wild-type embryos
(Fig. 2E,F). Furthermore, by
reducing the levels of chitin in wild-type embryos, which was achieved by
feeding wild-type parents with a low Nikkomycin dose (0.5 mg/ml), the
rtv tracheal phenotypes were reproduced
(Fig. 2G,H). These results thus
correlate with a requirement of Knk and Rtv in a linear pathway with
chitin.
The transient intraluminal chitin filament that assembles during the
restricted period of lumen expansion can be detected by labelling with a
FITC-conjugated chitin-binding probe (CBP)
(Tonning et al., 2005). CBP
labelling of wild type embryos reveals a luminal chitin fibre that is confined
to only part of the lumen and displays a filamentous texture
(Fig. 3A). In embryos produced
by wild-type parents fed with a high Nikkomycin dose (1 mg/ml) to reproduce
the kkv phenotype, luminal CBP levels are barely visible
(Fig. 3B). We used CBP to
assess luminal chitin levels in knk and rtv mutants, and
found that the tracheal lumen of both knk and rtv mutants
label with CBP. However, the luminal chitin in knk and rtv
mutants occupy their entire lumen and display an amorphous texture
(Fig. 3C,D).
We also noted that the intensity of the CBP labelling was reduced in
knk and rtv mutant trachea compared with wild type, which
may be due to the broader chitin distribution within the mutant lumens. Still,
in order to test if knk and rtv tracheal defects could be
due simply to reduced chitin levels, we contrasted the luminal CBP levels of
knk and rtv mutants with that of wild-type embryos treated
with a low Nikkomycin dose (0.5 mg/ml) to reproduce the rtv tracheal
phenotype. Interestingly, chitin diffuse to fill the lumen in embryos with
reduced chitin levels also (Fig.
3E), implying that proper chitin filament organization requires a
critical level of chitin synthesis. The intensity of the CBP-labelled chitin
in these embryos is, however, significantly weaker than in knk and
rtv mutant trachea (compare Fig.
3C-E). In addition, we find that Nikkomycin-treated embryos with
milder tracheal defects than that of knk and rtv mutants
also display weaker CBP staining than knk and rtv mutants
(not shown). Thus, Knk and Rtv appear required for chitin filament assembly,
rather than chitin synthesis. Another tracheal luminal filament that is
required for tracheal cell intercalation to form narrow tubes with
auto-cellular junctions contains the PioPio (Pio) protein
(Jazwinska et al., 2003).
However, the Pio filament is not affected in knk and rtv
mutants (Fig. 6M,N and not
shown), implying that Knk and Rtv are required for assembly of only a subset
of luminal matrix components.
|
), we
asked if knk mutants have a similar defect in the organization of the
chitinous procuticle. Cuticle preparations of mature knk embryos show
a dilated cuticle (Fig. 4A,B),
and the head skeleton and the pharynx of these embryos are deformed, as the
vertical plate is strongly melanized and appear granular instead of fibrous as
in the wild-type embryo (insets in Fig.
4A,B). These aspects of the knk cuticle are reminiscent
of that of kkv mutants (Ostrowski
et al., 2002; Moussian et al.,
2005a). TEM analysis of the knk mutant cuticle revealed
that the outermost layer of the stratified cuticle, the envelope, appear
normal in knk mutants, but that the space between the envelope and
the epidermal surface is filled with an amorphous substance containing
inclusions of electron-dense material, and the typical chitin lamellae
appeared absent (Fig. 4C,D).
Detection of cuticular chitin with gold-labelled Wheat germ agglutinin (WGA)
further show that WGA recognizes chitin in the knk mutant cuticle
(Fig. 4E,F), whereas WGA
labelling is absent in kkv mutants
(Fig. 4G). We also find that
the rtv mutant procuticle, which also lacks chitin lamellogenesis,
label with WGA (Fig. 4H). Thus,
the cuticle defects in knk and rtv mutants can be explained
by their specific requirement in chitin fibril formation.
Knk is an apical GPI-anchored protein that functions at the plasma membrane
The knk gene encodes a 689 amino acid protein with unknown
function (Ostrowski et al.,
2002). The protein is predicted to be extracellular and anchored
to the plasma membrane via a GPI moiety
(Fig. 5A). Knk also contains
two DM13 domains of unknown function and a DOMON domain predicted to form a
beta-sandwich structure that is seen in many extracellular adhesion domains,
including the fibronectin type III (fn3) domain
(Aravind, 2001).
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Consistent with a requirement during tracheal morphogenesis and later, for cuticle production, the knk mRNA is detected in the developing trachea from stage 13, just before tube expansion, and in the epidermis from late stage 15 (Fig. 5D-F). The Knk protein is also found in the developing trachea (Fig. 5G) and the late epidermis (Fig. 5I), but appear absent in knk mutants (Fig. 5H,K,L). Double labelling with anti-Knk and the septate junction marker Fas3 further show that Knk mainly localize to the apical plasma membrane of these cells, being present along the apical and apico-lateral cell surfaces, and partly overlapping with the distribution of the Fas3 protein (Fig. 5I,J).
In order to test if the function of Knk requires its membrane-bound form, rather than a cleaved form of the protein released into the lumen, we asked whether knk mutant embryos can be rescued by expression of a transmembrane Knk protein. We first established that ubiquitous expression of a UAS-knk transgene driven by Tubulin-GAL4 rescues both knk lethality and the tracheal and cuticle knk defects (Fig. 5M,N and not shown). We then replaced the predicted GPI anchor of Knk with the transmembrane portion of the Transferrin receptor (CG10620). Expression of the resulting UAS-knkTM in knk mutants using the same Tubulin-GAL4 driver also rescue both the knk tracheal phenotypes and lethality (Fig. 5O). Thus, membrane-bound Knk fulfils the requirements for Knk in tracheal tube expansion and cuticle deposition.
Knk is mislocalized in SJ mutants
The knk and rtv mutant phenotypes are also reminiscent of
that of mutants with disrupted SJ components (`SJ mutants')
(Beitel and Krasnow, 2000;
Behr et al., 2003;
Hemphala et al., 2003;
Paul et al., 2003;
Llimargas et al., 2004;
Wu et al., 2004). SJ mutants
display an overgrown DT at stage 15 with weak constrictions at fusion
branches, convoluted dorsal trunks at stage 16 and discontinuous GB lumens
(Beitel and Krasnow, 2000). As
the luminal chitinous matrix is disorganized in SJ mutants, it was suggested
that SJ components may function in tubulogenesis through their role in luminal
matrix assembly (Tonning et al.,
2005).
|
)
(Fig. 6G,H).
In order to further address the functional relationship between Knk and
SJs, we analysed double mutant combinations between knk and SJ
mutants. When chitin deficiency is combined with SJ mutations, the embryos
display a striking additive loss in relation to luminal 2A12 accumulation,
indicating that SJ proteins may be needed for the deposition or stability of
luminal components other than chitin
(Tonning et al., 2005). We
find that double mutants for knk and two SJ mutants, Fas2
and mega (pickel - FlyBase), also display an additive
reduction in luminal 2A12 levels (Fig.
6I-L and not shown), albeit not as strong as that observed in
Fas2;kkv and mega;kkv double mutants. These cellular and
genetic analyses further strengthen the idea that the requirement for Knk in
tube-size regulation is specifically related to that of chitin.
|
;
Hemphala et al., 2003)
(Fig. 7A), indicating that Grh
is not a prerequisite for developmental Knk expression. Knk was, however,
mislocalized in tube-expansion mutants, disrupting SJ components. In the five
SJ mutants analysed, Fas2, sinuous (sinu), mega,
bulbous (Lachesin - FlyBase) and Na pump 
subunit (ATP), Knk is distributed along the entire
lateral and basal cell surfaces instead of concentrating at the apical
tracheal epithelium (Fig. 7D-H
and not shown), Thus, SJ proteins appear to be required for correct targeting
of Knk to the apical surface, or to prevent its diffusion to other domains of
the plasma membrane. In order to assess whether this Knk mislocalization is
the cause for the tracheal tube size defects in SJ mutants, we tested if the
Fas2 and mega mutant luminal chitin matrix could be rescued
by ectopic expression of transmembrane Knk. Ectopic expression of
UAS-knkTM in Fas2 and mega mutant tracheal
epithelia was driven with the Btl-GAL4 driver-line, and was unable to restore
their chitin-filament structure and tube size defects
(Fig. 7I-L, and not shown).
These results thus suggest that additional components necessary for chitin
filament formation may be affected in SJ mutants.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chitin filament assembly
In chitin-containing extracellular matrices, the chitin chains often bundle
to form microfibrils. Such chitin chain bundling is mediated by hydrogen
bonding of amine and carbonyl groups between the single sugar chains, and
nascent chitin chains can spontaneously assemble into microfibrils. The insect
cuticle predominately contains -chitin, where ten or more polymers
assemble in an anti-parallel orientation stabilized by a high number of
hydrogen bonds to allow tight packaging
(Kramer and Koga, 1986;
Lehane, 1997;
Merzendorfer, 2005). The
fibrous appearance of the tracheal intraluminal matrix seen with CBP labelling
also probably represents bundles of chitin microfilaments. This is
strengthened by previous observations that Congo Red, a fluorescent dye that
intercalates between hydrogen-bonded chitin chains
(Cohen, 1993), visualizes
luminal chitinous filaments that run parallel with tube length
(Tonning et al., 2005). It is
unclear whether the tracheal luminal chitin also exhibits an -packaging
like the procuticle. Alternatively, the luminal chitin may assume a softer and
more flexible chitinous structure seen in the peritrophic matrix, in which the
chains are aligned in a parallel fashion (ß-form), involving fewer
inter-chain hydrogen bonds but increased hydrogen bonding with water molecules
that leads to its swelling (Peters,
1992; Merzendorfer,
2005).
Insect chitin synthases are large transmembrane proteins
(Tellam et al., 2000;
Merzendorfer and Zimoch,
2003), which produce and export chitin polymers to the
extracellular space. We find that decreased chitin synthesis upon treatment
with Nikkomycin alters the texture of the reduced luminal tracheal chitin, as
it fail to present the characteristic fibrillar structure. It is thus tempting
to speculate that a critical amount of localized chitin synthesis is required
for chitin-chain bundling. The glycosyltransferases that form the glycosidic
bonds in chitin, cellulose and hyaluronan belong to an evolutionarily
conserved family of proteins (Yoshida et
al., 2000; Coutinho et al.,
2003; Merzendorfer,
2005), and cellulose synthases are found to function as oligomers,
called rosettes, which are assembled in the Golgi and then transported to the
plasma membrane (Richmond and Somerville,
2000; Saxena et al.,
2001; Doblin et al.,
2002). Each rosette consists of six synthesizing units, and the
rosettes may align single cellulose chains before their spontaneous bundling
to yield a microfibril. In a similar way, chitin synthases could function only
after super-complex formation. If some of the chitin synthases in the complex
were inhibited by Nikkomycin, there would be fewer strands synthesized in each
complex that in turn could prevent microfilaments bundling.
The function of Knk and Rtv in chitin filament assembly
Based on genetic experiments and chitinous matrix analyses, we propose that
Knk and Rtv are specifically needed for chitin filament assembly. As Knk, and
possibly Rtv, associate with the external cell surface, and Knk can function
also as a transmembrane protein, the two proteins are not likely to be part of
the chitin matrices themselves. Instead, their requirements point to a role
for the epithelial cell surface in organizing the extracellular chitinous
matrix. No enzyme domains are detected in Knk and Rtv, and all the seven
knk mutant alleles characterized introduce non-sense mutations, which
is atypical for mutant alleles of genes that encode enzymes. It is thus likely
that Knk and Rtv are structural proteins assisting in chitin filament
assembly. Rtv exposes six aromatic residues that may bind to chitin
(Moussian et al., 2005b), and
the DOMON domain of Knk is predicted to form a Fibronectin type III-like (Fn3)
fold that mediates extracellular adhesion
(Aravind, 2001). Because chitin
synthases, as discussed above, may need to assemble into higher order
complexes to promote chitin microfibril formation, Knk and Rtv could assist in
correct CS-1 cluster formation and chitin chain assembly through interactions
with both CS-1 and chitin. Such a function may be analogous to that of Cobra,
a plant apical GPI-linked protein required for oriented deposition of
cellulose microfibrils and anisotropic cell expansion during plant
morphogenesis (Roudier et al.,
2005). Another possibility is that Knk and Rtv are needed for
higher-order chitin matrix assemblies. The yeast exocellular protein Gas1, for
example, is required for cell wall assembly and formation of cross-links
between the cell wall glucans, possibly by catalyzing a transglycosylation
reaction (Popolo and Vai,
1999). The localized activity of putative related enzymatic
modifications of Drosophila chitin matrices could thus depend on
apical Knk and Rtv. However, the enzyme Dopadecarboxylate, which is required
for synthesis of the important cuticle cross-linking quinones, is not required
for tracheal tube diameter expansion (not shown).
A role for tube expansion genes in chitin matrix formation
Several tracheal tube expansion mutants are identified that show various
degrees of tube diameter defects and overgrown tracheal tube lengths
(Beitel and Krasnow, 2000;
Hemphala et al., 2003;
Tonning et al., 2005). It
appears that most of these mutants affect the luminal chitin filament, which
is needed to coordinate tube diameter expansion and limit excess tube
elongation. Apart from kkv, encoding CS-1, the major group of
tube-expansion mutants disrupt SJ components and also affect luminal
components and chitin filament structure
(Wu and Beitel, 2004;
Tonning et al., 2005). As the
two new tube-expansion genes encoding Knk and Rtv appear specifically required
for the ordered assembly of chitin filaments into a functional fibrous matrix,
correct organization of the luminal chitin filament appears central for its
role in uniform tube diameter growth. Bundling of luminal chitin may enable
its uniform expansion upon lumen growth, which in turn promotes a uniform
expansion of the surrounding epithelium. Without properly bundled chitin, as
in mutants for knk, rtv and in SJ mutants, the chitinous matrix fills
the entire lumen and appears to have lost its rigidity, and the lumen swells
as if no or little matrix were present to shape the epithelium.
The mislocalization of Knk in mutants that disrupt SJ genes (Fas2,
bulb, sinu, ATP and mega), suggest that one role for SJs
in tracheal tube expansion is to ensure luminal chitin filament organization
through correct distribution of components involved in chitin filament
assembly. Knk mislocalization is, however, not sufficient to explain the
requirement for SJs in chitin matrix assembly, as overexpression of Knk in
Fas2 and mega mutants does not rescue the tracheal defects
in these mutants, and it is plausible that the proper localization of
additional components needed for chitin filament organization, for example
Rtv, also are affected in SJ mutants. Mutants for mega and
bulb display normal epithelial apico-basal polarity
(Behr et al., 2003;
Llimargas et al., 2004). Their
defects in polarized protein localization may thus expose a need for SJs in
protein export to the correct cell membrane domain, or in preventing diffusion
of proteins between the apical and baso-lateral compartments.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aravind, L. (2001). DOMON: an ancient extracellular domain in dopamine beta-monooxygenase and other proteins. Trends Biochem. Sci. 26,524 -526.[CrossRef][Medline]
Bakkers, J., Kramer, C., Pothof, J., Quaedvlieg, N. E., Spaink,
H. P. and Hammerschmidt, M. (2004). Has2 is required upstream
of Rac1 to govern dorsal migration of lateral cells during zebrafish
gastrulation. Development
131,525
-537.
Behr, M., Riedel, D. and Schuh, R. (2003). The claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila. Dev. Cell 5, 611-620.[CrossRef][Medline]
Beitel, G. J. and Krasnow, M. A. (2000). Genetic control of epithelial tube size in the Drosophila tracheal system. Development 127,3271 -3282.[Abstract]
Bray, S. J. and Kafatos, F. C. (1991).
Developmental function of Elf-1: an essential transcription factor during
embryogenesis in Drosophila. Genes Dev.
5,1672
-1683.
Cabib, E. (1991). Differential inhibition of
chitin synthetases 1 and 2 from Saccharomyces cerevisiae by polyoxin D and
nikkomycins. Antimicrob. Agents Chemother.
35,170
-173.
Cohen, E. (1993). Chitin synthesis and degradation as targets for pesticide action. Arch. Insect Biochem. Physiol. 22,245 -261.[CrossRef][Medline]
Cohen, E. (2001). Chitin synthesis and inhibition: a revisit. Pest Manag. Sci. 57,946 -950.[CrossRef][Medline]
Coutinho, P. M., Deleury, E., Davies, G. J. and Henrissat, B. (2003). An evolving hierarchical family classification for glycosyltransferases. J. Mol. Biol. 328,307 -317.[CrossRef][Medline]
Doblin, M. S., Kurek, I., Jacob-Wilk, D. and Delmer, D. P.
(2002). Cellulose biosynthesis in plants: from genes to rosettes.
Plant Cell Physiol. 43,1407
-1420.
Ghabrial, A., Luschnig, S., Metzstein, M. M. and Krasnow, M. A. (2003). Branching morphogenesis of the Drosophila tracheal system. Annu. Rev. Cell Dev. Biol. 19,623 -647.[CrossRef][Medline]
Grenningloh, G., Rehm, E. J. and Goodman, C. S. (1991). Genetic analysis of growth cone guidance in Drosophila: fasciclin II functions as a neuronal recognition molecule. Cell 67,45 -57.[CrossRef][Medline]
Hemphala, J., Uv, A., Cantera, R., Bray, S. and Samakovlis,
C. (2003). Grainy head controls apical membrane growth and
tube elongation in response to Branchless/FGF signalling.
Development 130,249
-258.
Jazwinska, A., Ribeiro, C. and Affolter, M. (2003). Epithelial tube morphogenesis during Drosophila tracheal development requires Piopio, a luminal ZP protein. Nat. Cell Biol. 5,895 -901.[CrossRef][Medline]
Jurgens, G., Wieschaus, E., Nusslein-Volhard, C. and Kluding, H. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. Roux Arch. Dev. Biol. 193,283 -295.[CrossRef]
Kramer, K. and Koga, D. (1986). Insect chitin: Physical state, synthesis, degradation and metabolic regulation. Insect Biochem. 16,851 -877.[CrossRef]
Lehane, M. J. (1997). Peritrophic matrix structure and function. Annu. Rev. Entomol. 42,525 -550.[CrossRef][Medline]
Llimargas, M., Strigini, M., Katidou, M., Karagogeos, D. and
Casanova, J. (2004). Lachesin is a component of a septate
junction-based mechanism that controls tube size and epithelial integrity in
the Drosophila tracheal system. Development
131,181
-190.
Merzendorfer, H. (2005). Insect chitin synthases: a review. J. Comp. Physiol. B,1 -15 [E-pub ahead of print].
Merzendorfer, H. and Zimoch, L. (2003). Chitin
metabolism in insects: structure, function and regulation of chitin synthases
and chitinases. J. Exp. Biol.
206,4393
-4412.
Moore, K. L. (2003). The biology and enzymology
of protein tyrosine O-sulfation. J. Biol. Chem.
278,24243
-24246.
Moussian, B., Schwarz, H., Bartoszewski, S. and Nusslein-Volhard, C. (2005a). Involvement of chitin in exoskeleton morphogenesis in Drosophila melanogaster. J. Morphol. 264,117 -130.[CrossRef][Medline]
Moussian, B., Soding, J., Schwarz, H. and Nusslein-Volhard, C. (2005b). Retroactive, a membrane-anchored extracellular protein related to vertebrate snake neurotoxin-like proteins, is required for cuticle organization in the larva of Drosophila melanogaster. Dev. Dyn. 233,1056 -1063.[CrossRef][Medline]
Ostrowski, S., Dierick, H. A. and Bejsovec, A.
(2002). Genetic control of cuticle formation during embryonic
development of Drosophila melanogaster. Genetics
161,171
-182.
Paul, S. M., Ternet, M., Salvaterra, P. M. and Beitel, G. J.
(2003). The Na+/K+ ATPase is required for septate junction
function and epithelial tube-size control in the Drosophila tracheal system.
Development 130,4963
-4974.
Peters, W. (1992). Peritrophic membranes: Zoophysiology Series, vol. 30. Berlin: Springer-Verlag.
Popolo, L. and Vai, M. (1999). The Gas1 glycoprotein, a putative wall polymer cross-linker. Biochim. Biophys. Acta 1426,385 -400.[Medline]
Richmond, T. A. and Somerville, C. R. (2000).
The cellulose synthase superfamily. Plant Physiol.
124,495
-498.
Roudier, F., Fernandez, A. G., Fujita, M., Himmelspach, R.,
Borner, G. H., Schindelman, G., Song, S., Baskin, T. I., Dupree, P.,
Wasteneys, G. O. et al. (2005). COBRA, an Arabidopsis
extracellular glycosyl-phosphatidyl inositol-anchored protein, specifically
controls highly anisotropic expansion through Its involvement in cellulose
microfibril orientation. Plant Cell
17,1749
-1763.
Samakovlis, C., Hacohen, N., Manning, G., Sutherland, D. C., Guillemin, K. and Krasnow, M. A. (1996). Development of the Drosophila tracheal system occurs by a series of morphologically distinct but genetically coupled branching events. Development 122,1395 -1407.[Abstract]
Saxena, I. M., Brown, R. M., Jr and Dandekar, T. (2001). Structure-function characterization of cellulose synthase: relationship to other glycosyltransferases. Phytochemistry 57,1135 -1148.[CrossRef][Medline]
Shiga, Y., Tanaka-Matakutsu, M. a. and Hayashi, S. (1996). A nuclear GFP/b-galactosidase fusion protein as a marker for morphogenesis in living Drosophila. Dev. Growth Differ. 38,99 -106.
Spicer, A. P. and Tien, J. Y. (2004). Hyaluronan and morphogenesis. Birth Defects Res. Part C Embryo Today 72,89 -108.[CrossRef][Medline]
Spradling, A. C., Stern, D., Beaton, A., Rhem, E. J., Laverty,
T., Mozden, N., Misra, S. and Rubin,G. M. (1999). The
Berkeley Drosophila genome project gene disruption project. Single P-element
insertions mutating 25% of vital Drosophila genes.
Genetics 153,135
-177.
Tellam, R. L., Vuocolo, T., Johnson, S. E., Jarmey, J. and Pearson, R. D. (2000). Insect chitin synthase cDNA sequence, gene organization and expression. Eur. J. Biochem. 267,6025 -6043.[Medline]
Tepass, U., Theres, C. and Knust, E. (1990). crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell 61,787 -799.[CrossRef][Medline]
Tonning, A., Hemphala, J., Tång, E., Nannmark, U., Samakovlis, C. and Uv, A. (2005). A transient luminal chitinous matrix is required to model epithelial tube diameter in the Drosophila trachea. Dev. Cell 3, 423-430.
Toole, B. P. (2004). Hyaluronan: from extracellular glue to pericellular cue. Nat. Rev. Cancer 4,528 -539.[CrossRef][Medline]
Uv, A., Cantera, R. and Samakovlis, C. (2003). Drosophila tracheal morphogenesis: intricate cellular solutions to basic plumbing problems. Trends Cell. Biol. 13,301 -309.[CrossRef][Medline]
Wieschaus, E. and Nüsslein-Volhard, C. (1986). Looking at embryos. In Drosophila: A Practical Approach (ed. D. B. Roberts), pp.199 -227. Oxford: IRL Press.
Wieschaus, E., Nusslein-Volhard, C. and Jurgens, G. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. Roux Arch. Dev. Biol. 193,196 -307.
Wu, V. M. and Beitel, G. J. (2004). A junctional problem of apical proportions: epithelial tube-size control by septate junctions in the Drosophila tracheal system. Curr. Opin. Cell Biol. 16,493 -499.[CrossRef][Medline]
Wu, V. M., Schulte, J., Hirschi, A., Tepass, U. and Beitel, G.
J. (2004). Sinuous is a Drosophila claudin required for
septate junction organization and epithelial tube size control. J.
Cell Biol. 164,313
-323.
Yoshida, M., Itano, N., Yamada, Y. and Kimata, K.
(2000). In vitro synthesis of hyaluronan by a single protein
derived from mouse HAS1 gene and characterization of amino acid residues
essential for the activity. J. Biol. Chem.
275,497
-506.
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