|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online December 20, 2005
doi: 10.1242/10.1242/dev.02206
1 Department of Medical Biochemistry, Göteborg University, Sweden.
2 Department of Genetics, Max-Planck-Institute for Developmental Biology,
Tübingen, Germany.
3 Microscopy Section, Max-Planck-Institute for Developmental Biology,
Tübingen, Germany.
* Author for correspondence (e-mail: bernard.moussian{at}tuebingen.mpg.de) and anne.uv{at}medkem.gu.se
Accepted 10 November 2005
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSImmunoblot analysisRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Chitin, Knickkopf, Apical ECM, Cuticle, Epidermis, Trachea, Mummy, Pyrophosphorylase, Udp-Glcnac, Ecdysone, Drosophila
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSImmunoblot analysisRESULTSDISCUSSIONREFERENCES |
|---|
; Jovine et al.,
2002; Roch et al.,
2003; Bökel et al.,
2005; Moussian and Uv,
2005). Apical ECMs are rich in carbohydrates such as
glucosaminoglycans and glycosyl-groups on proteins. These sugars are important
both for the polarised deposition of aECM components and their assembly into
functional aECMs. The glycosyl-groups bound up in aECMs originate from
cytosolic residues that are imported into the secretory compartments or used
directly by transmembrane enzymes to generate extracellular polysaccharides
(Wilson, 2002;
Huet et al., 2003;
Moore, 2003;
Mayor and Riezman, 2004). Many
enzymes acting in protein maturation are identified, but their contribution to
and requirements for aECM differentiation and organ morphogenesis in complex
multicellular organisms has been little investigated.
Differentiation of the Drosophila exoskeleton (cuticle), a
stratified highly ordered aECM, relies on a carefully orchestrated apparatus
for biosynthesis and modifications of proteins and the polysaccharide chitin.
The Drosophila cuticle is deposited during late embryogenesis to
cover the apical surface of the epidermis, as well as of the respiratory organ
(tracheae) and the fore- and hindgut
(Neville, 1975). The first
step in cuticle deposition is the formation of an outer impermeable envelope.
Subsequently, the middle protein-rich epicuticle responsible for cuticle
stiffness is formed and, finally, an innermost procuticle loaded with lamellar
linear chitin is assembled to confer stability and elasticity to the cuticle
(Locke, 2001).
Several Drosophila mutants have been identified that show defects
in cuticle formation without affecting the overall embryonic patterning
(Jürgens et al., 1984;
Nüsslein-Volhard et al.,
1984; Wieschaus et al.,
1984; Ostrowski et al.,
2002). One of these is krotzkopf verkehrt (kkv),
which encodes the epidermal chitin synthase CS-1. CS-1 is a transmembrane
enzyme that links cytosolic UDP-N-acetylglucosamine (UDP-GlcNAc) into long
GlcNAc polymers (chitin) that extrude from the apical surface. Loss of chitin
destabilises not only the procuticle, but also affects the integrity of the
epicuticle during cuticle differentiation suggesting that a proper procuticle
is required for epicuticle formation
(Moussian et al., 2005a). Loss
of function of two other factors, the predicted extracellular molecules
encoded by retroactive (rtv) and knickkopf
(knk), causes similar cuticle defects as mutations in kkv
and is required for chitin filament assembly
(Moussian et al., 2005b;
Moussian et al., 2006).
Another group of mutations that affects cuticle differentiation disrupt genes
that encode components of the septate junctions (SJs). SJs share functions
with vertebrate tight junctions, confer a paracellular diffusion barrier in
Drosophila epithelia (Lamb et
al., 1998; Wu and Beitel,
2004) and are necessary for polarised deposition of components
needed for cuticle assembly such as Knk
(Moussian et al., 2006). Both
groups of mutants additionally have impaired tracheal tube size regulation,
which is associated with a defective temporary chitin-containing luminal
matrix that is essential for uniform diameter growth
(Tonning et al., 2005).
We report on the phenotypic and molecular characterisation of
mummy (mmy), a mutation that is described to cause severe
cuticle defects (Nüsslein-Volhard et
al., 1984). The mmy mutant phenotype is similar to that
of the so-called `Halloween' mutants, which fail to produce the
differentiation hormone 20-Hydroxyecdysone
(Gilbert, 2004), and whose
role is still an enigma during insect embryogenesis. We find that mmy
codes for the Drosophila UDP-GlcNAc pyrophosphorylase that functions
in aECM formation by producing GlcNAc residues needed for chitin synthesis and
protein glycosylation, and that dynamic mmy expression is hormonally
regulated in aECM differentiating tissues.
|
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSImmunoblot analysisRESULTSDISCUSSIONREFERENCES |
|---|
; Bellen et al.,
2004). The deficiency uncovering the mmy locus and used
in this work is Df(2L)BSC6. Other mutants used were
shade7C (shd), shadow10D
(sad), kkv14C73 and kkvDZ8
(Jürgens et al., 1984).
Germline clones were generated using the Flp/FRT technique
(Chou et al., 1993). Tracheal
expression of UAS-Nod-lacZ (a chimeric construct of lacZ and
the microtubule minus-end motor Nod; provided by Yuh-Nung Jan) was driven by
Btl-Gal4, which expresses GAL4 in all tracheal cells from stage
12-13. The enhancer-trap marker 1-eve-1 (Tracheal-1) expresses
lacZ in all tracheal cells from tracheal cell invagination at stage
11 and has been described previously
(Samakovlis et al., 1996).
Transmission electron microscopy
For transmission electron microscopy (TEM), embryos were cryoimmobilised,
sectioned and contrasted as previously described
(Moussian et al., 2005a). For
gold-labelling of chitin, thin-sections of embryos were incubated with
biotinylated Wheat Germ Agglutinin (WGA; 1:500, Vector Laboratories) that was
recognised by an anti-biotin antibody (1:300, Enzo Diagnostics) that in turn
was detected by protein A conjugated to 10 nm gold particles (1:100, York
Stierhof). Gold-labelled specimens were contrasted for only 3 minutes instead
of 10 minutes.
Immunohistochemistry
Embryos were fixed and stained according to standard procedures. 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, MP), mouse IgG1 monoclonal anti-Crumbs (1:10; DSHB), mouse IgG
monoclonal anti-Discs large 1 (1:10; DSHB), mouse monoclonal IgG2a
anti-Fasciclin 3 (1:10; DSHB) and rabbit IgG polyclonal anti-Piopio (1:20; M.
Affolter). For fluorescent visualisation, the following secondary antibodies
were used: Alexa488 goat anti-rabbit IgG (1:500, MP), Alexa488 goat anti-mouse
IgM (1:500, MP), Alexa594 goat anti-mouse IgM (1:500, MP) and Alexa546 goat
anti-mouse IgG (1:500, MP). Labelling with fluorescein-conjugated
Chitin-binding probe (1:500, New England Biolabs, NEB) was performed according
to manufacturers recommendations. A BioRad Radiance 2000 was used to obtain
confocal images, and Nikon eclipse E1000 was used for obtaining fluorescent
images. Images were processed using Adobe Photoshop 7.0.
|
Immunoblot analysis |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSImmunoblot analysisRESULTSDISCUSSIONREFERENCES |
|---|
-tubulin (1:2000, Sigma). The primary antibodies were
detected with HRP-conjugated secondary antibodies. EndoH treatment was
performed according to the manufacturer (NEB).
In situ hybridisation
Whole-mount in situ hybridisation was performed with digoxigenin-labelled
RNA sense and antisense probes as described previously
(Tonning et al., 2005). RNA
probes for mmy were generated from the mmy cDNA LD24639 with
SP6 and T7 polymerases.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSImmunoblot analysisRESULTSDISCUSSIONREFERENCES |
|---|
). Compared with the wild-type larval cuticle
(Fig. 1A), the cuticle of
larvae harbouring the strong mmyIK63 allele is hardly
visible (Fig. 1B), whereas
larvae mutant for the weak mmyIL07 allele develop a
bloated cuticle and a deformed and strongly melanised head skeleton
(Fig. 1F,H).
In order to better understand the role of mmy in cuticle
differentiation, we compared the mmy mutant and wild-type larval
epidermis by transmission electron microscopy (TEM). Wild-type cuticle is
composed of three layers (Locke,
2001): the outermost envelope characterised by five alternating
electron-dense and electron-lucid sheets, the underlying epicuticle built up
by an upper electron-lucid and a lower electron-dense sublayer, and the
innermost procuticle structured by lamellar chitin microfibrils and contacting
the apical plasma membrane of the epidermal cells
(Fig. 2A). All three cuticle
layers are affected in mmyIK63 larvae
(Fig. 2B). The outer envelope
is thinner than in the wild type with only three sheets, and the
electron-dense sub-layer of the epicuticle disintegrates and spreads into the
upper electron-lucid sub-layer and the procuticle. The procuticle is also
reduced in thickness and seems to be devoid of chitin microfibrils;
occasionally, the cuticle detaches from the epidermal surface. The cuticle of
larvae mutant for the weak mmyIL07 allele is stratified as
in the wild type, and the procuticular chitin microfibrils appear correctly
oriented (Fig. 2C). However,
the mmyIL07 mutant procuticle contains abnormal inclusions
of electron-dense material that are scattered below the epicuticle, presumably
orphan proteins, suggesting that the coordinated assembly of the epi- and
procuticle is impaired. Taken together, cuticle assembly requires mmy
activity.
|
),
but on a specimen embedded in Epon and sectioned for TEM, WGA recognises only
chitin (Peters and Latka,
1986). Although chitin is present in wild-type and in the
mmyIL07 mutant procuticle
(Fig. 2D,F), no chitin is
detected in mutant larvae homozygous for the strong
mmyIK63 allele (Fig.
2E), arguing that mmy is essential for chitin synthesis.
However, mmy appears to have additional roles in cuticle
differentiation, as loss of chitin causes a compensatory increase in cuticular
protein deposition (Moussian et al.,
2005a), which is not detected in mmyIK63
mutants.
mmy is required for epithelial organisation
Our TEM analysis of the mmyIK63 cuticle also revealed
irregular cell shapes in the underlying epidermis. These cells are cuboidal,
rather than flattened like the wild-type epidermal cells
(Fig. 2A,B insets), and their
lateral membranes are not undulated. Epithelial cell shape is determined by
the cytoskeleton and cell-cell contacts. Using high resolution TEM analysis,
we find that the orientation of microtubules in wild-type,
mmyIL07 and mmyIK63 mutant larval
epidermal cells are indistinguishable (not shown), and that the adherens and
septate junctions (AJ and SJ) are positioned normally along the lateral
membrane (Tepass and Hartenstein,
1994). However, in the mmyIK63, but not
mmyIL07 mutant embryos, spacing between the epidermal
cells at the AJ appears wider than in the wild type (not shown), and the
characteristic ladder-like structure of the SJs is missing
(Fig. 3A-C). We also analysed
SJ integrity in the two mmy mutant epithelia, by labelling for the
two SJ components Fasciclin 3 (Fas3) and Discs large 1 (Dlg1). Both SJ
proteins are normally distributed in the weak mmyIL07
epithelia, and the intracellular Dlg1 protein is also normally localised in
the more severe mmyIK63 mutants (not shown). By contrast,
the transmembrane Fas3 is mislocalised along the lateral membrane in the
columnar epithelia of the hindgut and salivary gland in some
mmyIK63 embryos (Fig.
3D-G). Thus, the disrupted SJ ladder in
mmyIK63 mutants may reflect a requirement for Mmy in the
correct localisation of membrane-bound SJ components. As chitin-deficient
embryos have no detectable defects in SJ assembly or function
(Moussian et al., 2005a), and
chitin-deposition appears unaffected in embryos with disrupted SJ components
(Tonning et al., 2005), these
results point to two parallel requirements for mmy in chitin
synthesis and maturation of cell-cell contacts (SJs).
|
;
Ghabrial et al., 2003;
Uv et al., 2003;
Tonning et al., 2005). As
chitin is an essential component of this luminal matrix, we initially tested
whether mmy mutant embryos, which lack cuticular chitin, also display
tracheal tube size defects. The wild-type developing tracheal system can be
visualised with the lumen specific antibody 2A12
(Fig. 4A). The same staining
reveals a perfectly patterned tracheal system in embryos homozygous for the
weak mmyIL07 allele, although the dorsal trunk (DT) lumen
diameter is slightly irregular at stage 15
(Fig. 4B) and becomes
excessively elongated at stage 16 (not shown). The tracheal
mmyIK63 mutant lumen, however, fails to label with the
2A12-antibody, although the typical intracellular vesicular 2A12-staining is
present (Fig. 4C). Tracheal
tube shapes in mmyIK63 mutants were therefore visualised
with the pan-tracheal LacZ-enhancer-trap 1-eve-1. Detection
of the 1-eve-1 product shows that also mmyIK63
mutants display normal tracheal branch patterning, but tube diameter is
severely defective (Fig. 4D).
The mmyIK63 DT fusion branch lumens fail to expand and
remain constricted, whereas other parts of the DT trunk become excessively
dilated (Fig. 4E,F); during
stage 16, these DTs develop huge cyst-like structures
(Fig. 4G). Thus, mmy
is indeed required for uniform tube expansion.
The luminal tracheal chitin matrix can be detected both with a
FITC-conjugated chitin-binding probe (CBP) and WGA
(Tonning et al., 2005). CBP
appears specific for chitin and labels a broad filamentous chitin cable within
the wild-type tracheal lumen (Fig.
5A) that is absent in kkv mutant embryos
(Tonning et al., 2005). In
mutants for the weak mmyIL07 allele, CBP labels a luminal
cable with similar intensity to the wild type
(Fig. 5B), but its filamentous
appearance is slightly perturbed, implying that the chitin matrix is not
properly assembled. By contrast, embryos homozygous for the strong
mmyIK63 allele completely fail to label with CBP
(Fig. 5C), indicating a need
for mmy also in tracheal chitin synthesis. We also compared WGA
labelling of wild-type and mmyIK63 mutant embryos. In the
wild-type tracheae, WGA labels not only the broad luminal chitin cable, but
also the luminal surface that probably represents glycosylated proteins
(Fig. 5D). In the
mmyIK63 mutant tracheae, both the luminal and apical
WGA-staining is severely reduced (Fig.
5E). This is different from chitin-deficient embryos, where WGA
detects GlcNAc-groups along the apical cell surface and within the lumen,
although the broad luminal cable is absent
(Fig. 5F), arguing that
mmy function is required not only for chitin synthesis, but also for
other GlcNAc-containing components.
Despite the severe tracheal tube defects in mmyIK63
mutants, we find that apicobasal polarity appears normal. The transmembrane
protein Crumbs (Crb), which is required for cell polarisation and assembly of
the adherens junctions (AJs) (Tepass et
al., 1990), localises to the apical tracheal membrane in both
wild-type and mmyIK63 mutant embryos
(Fig. 5H,K), and the
microtubule minus end reporter, Nod:ß-gal
(Clark et al., 1997), appears
to accumulate correctly in the apical tracheal cell domain in the mutant
(Fig. 5I,L). Furthermore, no
significant defects in Fas3 localisation was observed in the
mmyIK63 mutant tracheal epithelium (not shown) and apical
secretion of the luminal protein Piopio (Pio), which is required to form
narrow tubes with autocellular junctions
(Jazwinska et al., 2003), was
normal in mmyIK63 mutants
(Fig. 5G,J). This indicates
that zygotic mmy is not generally required for apical protein
secretion.
We also observed that mmyIK63 mutants display severely
reduced WGA-labelling in embryonic tissues not known to produce chitin,
including the early epidermis and the salivary glands
(Fig. 5M-T). Moreover,
labelling for Crb revealed local tube dilations in the single-cell-layered
Malphigian tubules (both in the common ureter and in the individual tubules),
a failure of the salivary gland lumens to enlarge in late embryogenesis, and a
dorsal open phenotype in 
10 % of the mmy mutant embryos (not
shown), which may reflect a need for high levels of GlcNAc-containing
substances for these processes.
mmy encodes the Drosophila UDP-N-acetylglucosamine-pyrophosphorylase
In order to identify the gene disrupted in mmy mutants, we first
mapped the mutation to deficiency Df(2L)BSC6 on chromosome 2
(Fig. 1D). P-element induced
mutant fly strains known to segregate lethal mutations in this region were
subsequently tested in complementation crosses to the EMS-induced
mmyIK63 and mmyIL07 heterozygous
flies. Two P-element insertions isolated in the BDGP Gene Disruption project
(Bellen et al., 2004), KG08617
and KG04349, failed to complement the mmy EMS-induced mutations.
KG08617 fails to complement the lethality of both mmy EMS-alleles,
and embryos homozygous for this P-element insertion develop a cuticle
phenotype similar to homozygotes for mmyIK63
(Fig. 1C and not shown),
whereas KG04349 complements lethality, but the transhetereozygous flies are
sterile.
|
CG9535 is predicted to code for the Drosophila
UDP-N-acetylglucosamine-pyrophosphorylase (UDP-GlcNAc-Pyp), a cytosolic enzyme
catalyzing the formation of UDP-N-acetylglucosamine (UDP-GlcNAc) from UTP and
GlcNAc-1-Phosphate (Fig. 6C)
(Merzendorfer and Zimoch,
2003). CG9535 is the only predicted UDP-GlcNAc-Pyp in
Drosophila, and displays high amino acid homology to the
UDP-GlcNAc-Pyp in both yeast and humans (39% and 51% identity, respectively)
(Fig. 6B). As UDP-GlcNAc is the
substrate for chitin synthases
(Merzendorfer and Zimoch,
2003), a reduced level of UDP-GlcNAc in zygotic
mmyIK63 mutants would explain their chitin-deficiency in
the luminal tracheal matrix and in the cuticle, as well as a general loss of
GlcNAc-group in aECMs.
Protein modification is impaired in mmyIK63 embryos
Yeast and human UDP-GlcNAc-Pyp have been shown to exhibit also a
UDP-N-acetylgalactosamine (UDP-GalNAc)-Pyp activity
(Wang-Gillam et al., 2000;
Peneff et al., 2001).
Moreover, UDP-GlcNAc can be converted to UDP-GalNAc by UDP-N-acetylglucosamine
4-epimerase (Winans and Bertozzi,
2002; Mok et al.,
2005). Both UDP-GlcNAc and UDP-GalNAc are essential components of
the polysaccharide moieties that are added to secreted proteins upon
glycosylation and GPI-anchor synthesis in the ER and the Golgi apparatus.
In order to test whether mmy mutants display dysfunctional protein
maturation along the secretory pathway, we compared the size of the
extracellular cuticle protein Knickkopf (Knk)
(Jürgens et al., 1984;
Ostrowski et al., 2002) in
wild-type and mmy mutant larvae on Western blots
(Fig. 7A). The 689 amino acids
Knk protein is predicted to be N-glycosylated at three positions
(N170, N354 and N636), O-glycosylated at
S209 and T468, and to possess a GPI-anchor
(Mayor and Riezman, 2004;
Moussian et al., 2006). In
extracts of mmyIK63 and mmyKG08617
larvae, no wild-type sized Knk can be detected. Instead several proteins of
smaller molecular weight are recognised by the Knk antiserum, indicating that
post-translational modifications of Knk indeed are affected in the mutant
larvae. In addition, smaller molecular weight species of the plasma-membrane
protein Tout-velu (Ttv) (Bellaiche et al.,
1998), which is predicted to be N-glycosylated at N71,
N327 and N476, are present in mmy mutant larvae
when compared with the wild type, whereas no size changes are observed for the
intracellular membrane-bound Syntaxin1A
(Schulze and Bellen, 1996)
(Fig. 7B). Treatment of protein
extracts from wild-type larvae with EndoH, an enzyme that removes
N-glycosylated modification from proteins, produce Knk and Ttv proteins of
even higher mobility than those seen in mmy mutant larval extracts
(Fig. 7A,B), indicating that
protein glycosylation is not completely abolished in mmy mutant
larvae. By contrast, in protein extracts from mmyIL07
mutant larvae, which display strikingly similar cuticle phenotypes as
knk and rtv mutants
(Ostrowski et al., 2002;
Moussian et al., 2005b), and
mmyKG04349 mutant larvae, Knk and Ttv migration is not
detectably altered, indicating that protein modification in these mutant
backgrounds is not dramatically impaired.
To test whether impaired modification of Knk may affect its subcellular localisation, we analysed the distribution of Knk in the epidermis of stage 16 wild-type and mmyIK63 embryos labelled with the Knk antiserum (Fig. 7C,D). In the wild-type embryo, Knk is detected along the apical surface of the epidermal cell (Fig. 7C), whereas in mmyIK63 mutant embryos, only low levels of Knk signal are detected at the apical epidermal surface in addition to a faint cytoplasmic signal (Fig. 7D). This indicates that modification of Knk is required for its stability and localisation to the apical plasma membrane.
|
), and continues to be expressed at high levels in the
tracheae until stage 15 (Fig.
8D,E,G). This mid-embryonic mmy expression correlates
with luminal chitin deposition during tube expansion
(Tonning et al., 2005). At
stage 16, mmy is strongly expressed in the epithelium of the
developing salivary glands (Fig.
8F,H), which are not known to synthesise chitin, but may require
high levels of mmy to accommodate extensive protein secretion. First,
at late stage 16, and concurrent with chitin deposition, the mmy
transcript is detected in the epidermis
(Fig. 8F,I).
mmy expression is altered in mutants that disrupt 20-Hydroxyecdysone biosynthesis
The dynamic and tissue-specific zygotic expression of mmy may be
subjected to feedback regulation triggered by UDP-GlcNAc consumption during
aECM formation. However, a hypothetical accumulation or lack of UDP-GlcNAc in
kkv or mmyIK63 mutant embryos, respectively, did
not detectably alter the levels of mmy transcript, as mmy
was normally expressed in these mutants (not shown).
As mmy belongs to the group of Halloween mutants, including
shadow (sad) (Warren et
al., 2002) and shade (shd)
(Petryk et al., 2003), which
disrupt enzymes needed for biosynthesis of the insect hormone
20-hydroxyecdysone (Gilbert,
2004), we asked whether temporal expression of mmy in
different epithelia may depend on 20-hydroxyecdysone. The sad gene
encodes a mitochondrial P450 enzyme (CYP315A1), a C2-hydroxylase that
catalyses the formation of ecdysone from 2-deoxyecdysone; shd encodes
the enzyme CYP314A1, which is responsible mono-oxygenation of ecdysone to
generate the active 20-hydroxyecdysone. Indeed, two aspects of mmy
expression are similarly altered in embryos mutant for sad and
shd. First, we find that shd and sad mutants lack
the mid-embryonic up-regulation of tracheal mmy expression, and
second, mmy expression is prematurely upregulated in the epidermis,
salivary gland and proventriculus (Fig.
8J-O). Thus, a mid-embryonic `ecdysone pulse' appears essential to
control the temporal expression of mmy in different embryonic
epithelia.
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSImmunoblot analysisRESULTSDISCUSSIONREFERENCES |
|---|
mmy encodes the Drosophila UDP-N-acetylglucosamine-pyrophosphorylase
Formation of UDP-GlcNAc from GlcNAc-6-phosphate and UTP has been
demonstrated in vitro for the yeast protein UDP-GlcNAc-Pyp (UAP1)
(Mio et al., 1998). Yeast
cells lacking UAP1 activity have an abnormal bloated cell shape indicative of
a weakened cell wall. The human orthologue of UAP1 (AGX) was identified in the
same study and was able to rescue the cell lethal phenotype of UAP1
mutants, demonstrating a conserved function for UDP-GlcNAc-Pyp among species.
Mmy (CG9535) is the predicted Drosophila orthologue of yeast and
human UDP-GlcNAc-Pyp, and is thus expected to be responsible for UDP-GlcNAc
synthesis in Drosophila. UDP-GlcNAc-Pyp from different species have
also been shown to have UDP-GalNAc-Pyp activity
(Szumilo et al., 1996;
Peneff et al., 2001). In
addition, UDP-GlcNAc is converted to UDP-GalNAc by the
UDP-N-acetylglucosamine 4-epimerase
(Winans and Bertozzi, 2002).
Hence, Drosophila Mmy may also be responsible for UDP-GalNAc
production.
The EMS induced mutations mmyIK63 and
mmyIL07 identify important amino acids for Mmy activity.
Gly261 that is exchanged to valine in the MmyIK63
protein corresponds to Gly224 in the human AGX that has been shown
to be essential for enzyme activity by site-directed mutagenesis and lies in a
loop contacting uridine, as demonstrated by crystallographic experiments
(Wang-Gillam et al., 2000;
Peneff et al., 2001). As the
phenotypes caused by the exchange of Gly261 and by the deletion of
mmy in our P-element excision-lines are identical, it is conceivable
that this mutation causes a loss of Mmy function. The highly conserved
Ile197 that is mutated in the mmyIL07 allele
has not yet been described to be essential for UDP-GlcNAc-Pyp activity. This
amino acid exchange permits the production of some UDP-GlcNAc, as many
processes that require zygotic Mmy activity appear normal in
mmyIL07 embryos.
|
;
Moussian et al., 2006). We
find that Knk is a target of Mmy activity, as embryos homozygous for the
amorphic alleles mmyIK63 and
mmyKG08617 produce Knk proteins with reduced molecular
weight, compared with wild-type Knk, indicative of impaired glycosylation,
obviously affecting its localisation and function. However, paired Knk
function in combination with lack of chitin is not sufficient to explain fully
the aECM defects in mmy mutants, as loss of Knk and chitin cause
similar phenotypes to loss of chitin only
(Ostrowski et al., 2002;
Moussian et al., 2006). The
observed requirement of zygotic Mmy for Knk modification instead suggests that
additional proteins needed for cuticle differentiation and tracheal luminal
matrix formation, such as Retroactive (Rtv)
(Moussian et al., 2005b), are
affected in mmy mutants.
The disrupted aECMs in mmy mutants may also be contributed to by
disorganised aECM-producing epithelia. Although zygotic Mmy is not required
for epithelial apicobasal polarity, we find that SJ in mmy mutant
epithelia fail to form the typical ladder-like structure, which occasionally
is accompanied by slight defects in Fas3 localisation at these junctions.
Mutants lacking single SJ components display defects both in tracheal luminal
matrix formation and cuticle deposition
(Llimargas, 2000;
Behr et al., 2003;
Wu et al., 2004). This role of
SJ components may be, at least partially, mediated through correct
localisation of the cuticle organising Knk protein
(Moussian et al., 2006).
Moreover, the tracheal lumen of embryos deficient for both chitin and single
SJ components fails to label with the 2A12 antibody, as seen in mmy
mutants (Tonning et al.,
2005). Thus, mmy may also affect the aECM through its
need for SJ assembly. Given the requirements for mmy in protein
modification along the secretory pathway, Mmy is likely to affect primarily
the localised deposition and function of extracellular SJ proteins.
|
). In
addition, transcription control of chitin synthase and components of the
hexosamine pathway occurs in the mosquito midgut epithelium when the apical
chitin-containing peritrophic matrix is altered upon contact with a blood meal
(Lemos et al., 1996;
Smartt et al., 1998;
Ibrahim et al., 2000). We find
that mmy is regulated also at the transcriptional level in that it is
strikingly upregulated in different epithelia at the time they produce aECMs.
High levels of mmy transcript, for example, are limited first to the
period of luminal chitin accumulation during tube expansion and later turns on
when cuticle deposition begins.
Our data indicate that mmy expression is under the control of the
insect steroid hormone 20-hydroxyecdysone. There are several peaks of
20-hydroxyecdysone levels during insect development, and the role of these
peaks during larval moults and metamorphosis are well studied
(Thummel, 2001). In addition,
20-hydroxyecdysone levels peak at mid-embryogenesis, but the cellular
mechanisms activated by this rise of 20-hydroxyecdysone have been unclear.
Phenotypically, mmy belongs to the so-called Halloween mutants
(Chavez et al., 2000), which
develop a faint cuticle including shadow (sad) and
shade (shd)
(Jürgens et al., 1984;
Nüsslein-Volhard et al.,
1984) coding for 20-hydroxyecdysone-producing enzymes
(Gilbert, 2004). Unlike
sad and shd, mmy mutant embryos can activate the
20-hydroxyecdysone response element (Chavez
et al., 2000), suggesting that zygotic mmy itself is not
a component of ecdysone signalling pathway. Here, we show that loss of
20-hydroxyecdysone in sad and shd mutant embryos prevents
the strong tracheal mmy expression normally seen from stage 12 to 15.
These results indicate that a function of the mid-embryogenic pulse of
20-hydroxyecdysone is to activate a programme for aECM differentiation in the
developing tracheae. To date, we cannot explain the implications of the
premature upregulation of mmy in the epidermis and the salivary
glands of 20-hydroxyecdysone deficient embryos; however, 20-hydroxyecdysone
levels appear important for early epidermal morphogenesis as sad and
shd mutant embryos fail to undergo head involution and dorsal
closure. The temporal onset of mmy expression to accommodate
sufficient UDP-GlcNAc/GalNAc for aECM formation thus appears ensured by a
composite mmy promoter that can positively and negatively respond to
20-hydroxyecdysone, depending on tissue type and developmental time frame.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSImmunoblot analysisRESULTSDISCUSSIONREFERENCES |
|---|
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]
Bellaiche, Y., The, I. and Perrimon, N. (1998). Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion. Nature 394, 85-88.[CrossRef][Medline]
Bellen, H. J., Levis, R. W., Liao, G., He, Y., Carlson, J. W.,
Tsang, G., Evans-Holm, M., Hiesinger, P. R., Schulze, K. L., Rubin, G. M. et
al. (2004). The BDGP gene disruption project: single
transposon insertions associated with 40% of Drosophila genes.
Genetics 167,761
-781.
Bulik, D. A., van Ophem, P., Manning, J. M., Shen, Z., Newburg,
D. S. and Jarroll, E. L. (2000). UDP-N-acetylglucosamine
pyrophosphorylase, a key enzyme in encysting Giardia, is allosterically
regulated. J. Biol. Chem.
275,14722
-14728.
Bökel, C., Prokop, A. and Brown, N. H.
(2005). Papillote and Piopio: Drosophila ZP-domain proteins
required for cell adhesion to the apical extracellular matrix and microtubule
organization. J. Cell Sci.
118,633
-642.
Chavez, V. M., Marques, G., Delbecque, J. P., Kobayashi, K., Hollingsworth, M., Burr, J., Natzle, J. E. and O'Connor, M. B. (2000). The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels. Development 127,4115 -4126.[Abstract]
Chou, T.-B., Noll, E. and Perrimon, N. (1993). Autosomal P[ovoD1] dominant female-sterile insertions in Drosophila and their use in generating germ-line chimeras. Development 119,1359 -1369.[Abstract]
Clark, I. E., Jan, L. Y. and Jan, Y. N. (1997). Reciprocal localization of Nod and kinesin fusion proteins indicates microtubule polarity in the Drosophila oocyte, epithelium, neuron and muscle. Development 124,461 -470.[Abstract]
D'Amico, P. and Jacobs, J. R. (1995). Lectin histochemistry of the Drosophila embryo. Tissue Cell 27, 23-30.[CrossRef][Medline]
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]
Gilbert, L. I. (2004). Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster. Mol. Cell Endocrinol. 215, 1-10.[CrossRef][Medline]
Huet, G., Gouyer, V., Delacour, D., Richet, C., Zanetta, J. P., Delannoy, P. and Degand, P. (2003). Involvement of glycosylation in the intracellular trafficking of glycoproteins in polarized epithelial cells. Biochimie 85,323 -330.[Medline]
Ibrahim, G. H., Smartt, C. T., Kiley, L. M. and Christensen, B. M. (2000). Cloning and characterization of a chitin synthase cDNA from the mosquito Aedes aegypti. Insect Biochem. Mol. Biol. 30,1213 -1222.[CrossRef][Medline]
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]
Jovine, L., Qi, H., Williams, Z., Litscher, E. and Wassarman, P. M. (2002). The ZP domain is a conserved module for polymerization of extracellular proteins. Nat. Cell Biol. 4,457 -461.[CrossRef][Medline]
Jürgens, 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]
Lamb, R. S., Ward, R. E., Schweizer, L. and Fehon, R. G.
(1998). Drosophila coracle, a member of the protein 4.1
superfamily, has essential structural functions in the septate junctions and
developmental functions in embryonic and adult epithelial cells.
Mol. Biol. Cell 9,3505
-3519.
Lemos, F. J., Cornel, A. J. and Jacobs-Lorena, M. (1996). Trypsin and aminopeptidase gene expression is affected by age and food composition in Anopheles gambiae. Insect Biochem. Mol. Biol. 26,651 -658.[CrossRef][Medline]
Llimargas, M. (2000). Wingless and its signalling pathway have common and separable functions during tracheal development. Development 127,4407 -4417.[Abstract]
Locke, M. (2001). The Wigglesworth Lecture: Insects for studying fundamental problems in biology. J. Insect Physiol. 47,495 -507.[CrossRef][Medline]
Manning, G. and Krasnow, M. A. (1993). Development of the Drosophila tracheal system. In The Development of Drosophila melanogaster (ed. A. Martinez-Arias and M. Bate), pp. 609-686. New York: Cold Spring Harbor Laboratory Press.
Mayor, S. and Riezman, H. (2004). Sorting GPI-anchored proteins. Nat. Rev. Mol. Cell. Biol. 5, 110-120.[CrossRef][Medline]
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.
Mio, T., Yabe, T., Arisawa, M. and Yamada-Okabe, H.
(1998). The eukaryotic UDP-N-acetylglucosamine
pyrophosphorylases. Gene cloning, protein expression, and catalytic mechanism.
J. Biol. Chem. 273,14392
-14397.
Mok, M. T., Tay, E., Sekyere, E., Glenn, W. K., Bagnara, A. S. and Edwards, M. R. (2005). Giardia intestinalis: Molecular characterization of UDP-N-acetylglucosamine pyrophosphorylase. Gene 357,73 -82.[CrossRef][Medline]
Moore, K. L. (2003). The biology and enzymology
of protein tyrosine O-sulfation. J. Biol. Chem.
278,24243
-24246.
Moussian, B. and Uv, A. E. (2005). An ancient control. of epithelial barrier formation and wound healing. BioEssays 27,987 -990.[CrossRef][Medline]
Moussian, B., Schwarz, H., Bartoszewski, S. and Nüsslein-Volhard, C. (2005a). Involvement of chitin in exoskeleton morphogenesis in Drosophila melanogaster. J. Morphol. 264,117 -130.[CrossRef][Medline]
Moussian, B., Söding, J., Schwarz, H. and Nüsslein-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]
Moussian, B., Tång, E., Tonning, A., Helms, S., Schwartz,
H., Nüsslein-Volhard, C. and Uv, A. E. (2006).
Drosophila Knickkopf and Retroactive are needed for epithelial tube growth and
cuticle differentiation through their specific requirement for chitin filament
organization. Development
133,163
-171.
Neville, A. C. (1975). Biology of the Arthropod Cuticle. Berlin, Heidelberg, New York: Springer-Verlag.
Nüsslein-Volhard, C., Wieschaus, E. and Kluding, H. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. Roux Arch. Dev. Biol. 193,267 -282.[CrossRef]
Ostrowski, S., Dierick, H. A. and Bejsovec, A.
(2002). Genetic control of cuticle formation during embryonic
development of Drosophila melanogaster. Genetics
161,171
-182.
Peneff, C., Ferrari, P., Charrier, V., Taburet, Y., Monnier, C., Zamboni, V., Winter, J., Harnois, M., Fassy, F. and Bourne, Y. (2001). Crystal structures of two human pyrophosphorylase isoforms in complexes with UDPGlc(Gal)NAc: role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture. EMBO J. 20,6191 -6202.[CrossRef][Medline]
Peters, W. and Latka, I. (1986). Electron microscopic localization of chitin using colloidal gold labelled with wheat germ agglutinin. Histochemistry 84,155 -160.[CrossRef][Medline]
Petryk, A., Warren, J. T., Marques, G., Jarcho, M. P., Gilbert,
L. I., Kahler, J., Parvy, J. P., Li, Y., Dauphin-Villemant, C. and O'Connor,
M. B. (2003). Shade is the Drosophila P450 enzyme that
mediates the hydroxylation of ecdysone to the steroid insect molting hormone
20-hydroxyecdysone. Proc. Natl. Acad. Sci. USA
100,13773
-13778.
Roch, F., Alonso, C. R. and Akam, M. (2003).
Drosophila miniature and dusky encode ZP proteins required for cytoskeletal
reorganisation during wing morphogenesis. J. Cell Sci.
116,1199
-1207.
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]
Schulze, K. L. and Bellen, H. J. (1996). Drosophila syntaxin is required for cell viability and may function in membrane formation and stabilization. Genetics 144,1713 -1724.[Abstract]
Sebastiano, M., Lassandro, F. and Bazzicalupo, P. (1991). cut-1 a Caenorhabditis elegans gene coding for a dauer-specific noncollagenous component of the cuticle. Dev. Biol. 146,519 -530.[CrossRef][Medline]
Smartt, C. T., Chiles, J., Lowenberger, C. and Christensen, B. M. (1998). Biochemical analysis of a blood meal-induced Aedes aegypti glutamine synthetase gene. Insect Biochem. Mol. Biol. 28,935 -945.[CrossRef][Medline]
Szumilo, T., Zeng, Y., Pastuszak, I., Drake, R., Szumilo, H. and
Elbein, A. D. (1996). Purification to homogeneity and
properties of UDP-GlcNAc (GalNAc) pyrophosphorylase. J. Biol.
Chem. 271,13147
-13154.
Tepass, U. and Hartenstein, V. (1994). The development of cellular junctions in the Drosophila embryo. Dev. Biol. 161,563 -596.[CrossRef][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]
Thummel, C. S. (2001). Molecular mechanisms of developmental timing in C. elegans and Drosophila. Dev. Cell 1,453 -465.[CrossRef][Medline]
Tonning, A., Hemphala, J., Tang, 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 9,423 -430.[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]
Wang-Gillam, A., Pastuszak, I., Stewart, M., Drake, R. R. and
Elbein, A. D. (2000). Identification and modification of the
uridine-binding site of the UDP-GalNAc (GlcNAc) pyrophosphorylase.
J. Biol. Chem. 275,1433
-1438.
Warren, J. T., Petryk, A., Marques, G., Jarcho, M., Parvy, J.
P., Dauphin-Villemant, C., O'Connor, M. B. and Gilbert, L. I.
(2002). Molecular and biochemical characterization of two P450
enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster.
Proc. Natl. Acad. Sci. USA
99,11043
-11048.
Wieschaus, E., Nüsslein-Volhard, C. and Jürgens, G. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. III. Zygotic loci on the X-chromosome and fourth chromosome. Roux Arch. Dev. Biol. 193,296 -307.[CrossRef]
Wilson, I. B. (2002). Glycosylation of proteins in plants and invertebrates. Curr. Opin. Struct. Biol. 12,569 -577.[CrossRef][Medline]
Winans, K. A. and Bertozzi, C. R. (2002). An inhibitor of the human UDP-GlcNAc 4-epimerase identified from a uridine-based library: a strategy to inhibit O-linked glycosylation. Chem. Biol. 9,113 -129.[CrossRef][Medline]
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., Tepas
