|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 16 August 2006
doi: 10.1242/dev.02525
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SG1 and PH4SG2
Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2196, USA.
Author for correspondence (e-mail:
dandrew{at}jhmi.edu)
Accepted 7 July 2006
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
SG1 and PH4SG2. We show
through in vitro DNA-binding studies and in vivo expression assays that Fkh
cooperates with the salivary gland-specific bHLH protein Sage to directly
regulate expression of PH4SG2, as well as
sage itself, and to indirectly regulate expression of
PH4SG1. PH4SG1 and
PH4SG2 encode -subunits of resident ER enzymes
that hydroxylate prolines in collagen and other secreted proteins. We
demonstrate that salivary gland secretions are altered in embryos missing
function of PH4SG1 and
PH4SG2; secretory content is reduced and shows
increased electron density by TEM. Interestingly, the altered secretory
content results in regions of tube dilation and constriction, with
intermittent tube closure. The regulation studies and phenotypic
characterization of PH4SG1 and
PH4SG2 link Fkh, which initiates tube formation, to
the maintenance of an open and uniformly sized secretory tube.
Key words: Drosophila, Fork head (Fkh), Prolyl-4-hydroxylase, Sage, Salivary gland, Tube morphogenesis
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Bradley et al., 2001;
Abrams et al., 2003). Salivary
gland formation requires the homeotic transcription factors Sex combs reduced
(Scr), Extradenticle (Exd) and Homothorax (Hth); in loss-of-function mutants
for any one of the corresponding genes, salivary glands fail to form.
Moreover, ectopic expression of Scr leads to formation of additional salivary
glands (Panzer et al., 1992;
Andrew et al., 1994).
Expression of Scr and hth and nuclear localization of Exd
disappear shortly after the gland initiates morphogenesis
(Henderson and Andrew, 2000),
suggesting that genes specifying the salivary gland fate are not directly
involved in terminal differentiation.
Several transcription factor genes are expressed in the early salivary
gland under the control of Scr, Exd and Hth, including fkh, which
encodes a Fox family winged-helix transcription factor homologous to mammalian
Foxa2 (HNF3ß) and C. elegans PHA-4
(Weigel et al., 1989a;
Weigel et al., 1989b;
Horner et al., 1998;
Kalb et al., 1998). In the
embryo, Fkh controls apical constriction of the salivary cells as they
invaginate to form tubes and promotes salivary cell survival by inhibiting
apoptosis (Myat and Andrew,
2000). Fkh also prevents the expression of duct-specific genes in
the secretory region of the gland (Kuo et
al., 1996; Haberman et al.,
2003), maintains its own expression
(Zhou et al., 2001) and
maintains expression of CrebA, a bZip transcription factor required
to upregulate expression of secretory pathway component genes
(Abrams and Andrew, 2005). In
prepupal larvae, Fkh activates expression of the sgs glue genes,
which encode secreted proteins that allow the pupae to adhere to a substratum
(Lehmann and Korge, 1996;
Mach et al., 1996). Thus, Fkh
is required throughout gland development.
To learn more about the range of activities Fkh has in the salivary gland
and to understand the mechanisms of Fkh gene regulation, we identified and
characterized two downstream targets with Fkh-dependent expression:
PH4SG1 and PH4SG2, referred
to as SG1 and SG2. We show that Fkh works with Sage, a
salivary gland-specific bHLH protein, to regulate expression of SG2
directly as well as expression of sage itself. Surprisingly, we show
that although SG1 is also regulated by Fkh and Sage, its regulation
by these proteins is indirect.
SG1 and SG2 encode homologues of the -subunits of
ER enzymes that hydroxylate proline residues in collagen, a major component of
the extracellular matrix, as well as other secreted proteins
(Kivirikko and Pihlajaniemi,
1998). As the Drosophila collagen genes are not expressed
in the embryonic salivary gland (Le Parco
et al., 1986; Knibiehler et
al., 1990; Yasothornsrikul et
al., 1997; Chartier et al.,
2002), these enzymes must hydroxylate proline residues in other
secreted or transmembrane proteins. Consistent with this idea, apical salivary
gland secretions were altered in embryos missing SG1 and
SG2; secretory content was reduced in volume, was more electron dense
and was less fibrillar when assessed by transmission electron microscopy. The
loss of secretory volume correlated with regions of tube dilation,
constriction and closure.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). Immunohistochemistry was carried out using
Biotin-conjugated horse-radish peroxidase (HRP) for light microscopy
(Reuter et al., 1990) and the
following primary antibodies: SG1 (1:15,000, this work), SG2
(1:8,000, this work), ßgal (1:5000; Promega; Madison, WI) and
Fkh (1:1000). Biotin-conjugated secondary antibodies were used at a
dilution of 1:500 and signal was amplified using the Vectastain kit (Vector
Laboratories; Burlingame, CA). ß-gal staining of all reporter constructs
from the same gene were carried out at the same time with approximately the
same volume of embryos (
100 µl) and were reacted for exactly 3 minutes
to prevent variations in signal resulting from differences in embryo volume,
antibody variation and reaction time differences. Immunohistochemistry for
confocal microscopy was carried out using the following antibodies:
KDEL (1:500; Stressgen, San Diego, CA), SG1 (1:5,000),
SG2 (1:1,000), ßgal (1:500; Promega; Madison, WI),
Crb [1:30; Drosophila Hybridoma Studies Bank (DHSB); Iowa City, Iowa],
CrebA (1:500) (Andrew et al.,
1997), Spectrin (1:1; DHSB),
ßHspectrin (1:100)
(Thomas and Kiehart, 1994) and
Catenin (1:250) (Oda et al.,
1994). Fluorescently tagged Alexa-secondary antibodies were used
at a dilution of 1:500 (Molecular Probes; Eugene, OR).
Antiserum production
Antiserum was generated in rat to an N-terminal fragment of SG1 and in
rabbit to an N-terminal fragment of SG2 (Covance; Denver, PA). The details of
cloning, protein production and purification are available upon request.
Fkh protein purification and electrophoretic gel mobility shift assays
The region encoding the DNA-binding domain (DBD) of Fkh was amplified from
genomic DNA by PCR and subcloned into the expression vector pProEx
(Invitrogen; Carlsbad, CA). Recombinant protein was induced by the same method
used for antiserum production (contact authors for details) and purified under
denaturing conditions by affinity purification with Ni-NTA agarose beads,
according to the manufacturer's protocol (Qiagen, Germany). The resulting
protein preparation, which was reasonably pure (a single visible band on
Coomassie stained SDS-PAGE), was concentrated 10x using a Vivaspin
concentrator (Vivascience; Hannover, Germany) to a final concentration of 1
µg/µl and stored at 4°C until use.
Plus- and minus-strand oligonucleotides corresponding to the putative Fkh-binding sites upstream of SG1 and SG2 (see Figs 2 and 3 for sequences) were synthesized by the Johns Hopkins Biosynthesis and Sequencing Facility. The plus-strand oligos were kinase-labeled according to the manufacturer's protocol (Invitrogen; Carlsbad, CA) and annealed to the corresponding minus strand by boiling the samples for 5 minutes and cooling them to room temperature. Unlabelled double-stranded oligos were similarly prepared for competition experiments. Binding reactions were carried out in DNA-binding buffer (20 mM HEPES, pH 6.8; 40 mM KCl), 1 mM DTT, 10% glycerol, 2 µg of Fkh DBD protein and 11.9 pmol of labeled oligonucleotide probe. Poly dIdC (1.4 µg) was added as a nonspecific binder to each reaction. Cold competitor oligos were added last (at 15x, 30x and 60x the concentration of the labeled oligos) and the reactions were incubated for 20 minutes on ice. Binding reactions were run on 7% polyacrylamide gels (50 mM HEPES pH 6.8) for 6 hours at 10 mA at 4°C and prepared for autoradiography by standard methods.
Fly strains
fkh6 H99 (Myat and
Andrew, 2000) and fkhP261 (M. M. Myat and
D.J.A., unpublished) were used for fkh regulation studies.
Df(3R)Exel6216 was generated by Exelixus
(Parks et al., 2004). Mutant
and deficiency lines were maintained over a third chromosome balancer
(TM3 or TM6B) carrying Ubx-lacZ or
twi-GFP constructs to allow identification of homozygous
mutants by the absence of lacZ hybridization, ßgal staining or
GFP fluorescence. fkh-GAL4
(Henderson and Andrew, 2000)
or tubulin-GAL4 (Lee and Luo,
1999) were used to drive expression of UAS constructs.
Generation of reporter gene and expression constructs
The details for subcloning the open reading frames (ORF) for sage,
SG1 and SG2 into pUAST (Brand
and Perrimon, 1993), and for subcloning the salivary gland
enhancers for all three genes into the Casper ß-gal vector
(Thummel et al., 1988) are
available upon request. Mutations in candidate binding sites were made by site
directed mutagenesis using the QuikChange XL Kit or QuikChange Multi
Site-Directed Mutagenesis Kit (Stratagene; Cedar Creek, TX).
The reporter gene and expression constructs were introduced into the
germline of w1118 flies
(Spradling and Rubin, 1982).
The insertions were mapped to individual chromosomes genetically by following
the w+ eye color marker using marked balancer
chromosomes.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
subunits, six of which showed tissue-specific embryonic
expression (Abrams and Andrew,
2002). Two of the genes, SG1 and SG2, were
specifically expressed in the salivary glands beginning at stage 11 and
continuing through embryogenesis and larval stages
(Fig. 1A, B; data not shown).
To learn which early transcription factors regulate SG1 and
SG2, we examined expression of the two genes in embryos mutant for
the salivary gland genes, fkh, CrebA and huckebein
(hkb). Although CrebA and hkb had no effect on
expression, SG1 and SG2 mRNA disappeared in fkh
mutants (Fig. 1A,B; data not
shown). As with the mRNAs, SG1 and SG2 proteins, which localize to the ER
(Fig. 1E), were not detected in
the fkh mutants (Fig.
1D; data not shown). Thus, salivary gland expression of
SG1 and SG2 requires Fkh.
To begin to identify sequences required for SG1 and SG2
expression, we generated transgenic lines carrying reporter gene constructs
with 1 kb of upstream DNA, including a small region of the 5' end
of the ORF, fused to lacZ (Fig.
2A and
3A; SG1 972-lacZ and
SG2 885-lacZ). As with the endogenous genes, lacZ expression
(measured by ßgal staining) from all of the transgenic lines carrying
each reporter gene construct was observed in the salivary gland beginning at
stage 11 and continuing through embryogenesis
(Fig. 2B-D;
Fig. 3B-D). Variable expression
of ßgal was also observed in other cells; for example, expression of
ßgal was seen in the embryonic hemocytes with one SG1 972-lacZ
line (Fig. 2B-D,H,I) and in a
subset of gut endodermal cells with all the SG2 885-lacZ lines
(Fig. 3B-D,H). Salivary gland
staining for both the SG1 and SG2 constructs disappeared in
fkh mutants (Fig. 2I,
Fig. 3I), although the
hemocyte-specific staining with the SG1 972-lacZ line was still
observed (Fig. 2I).
To test whether Fkh regulation of SG1 and SG2 is direct,
we first asked if Fkh binds in vitro to the sequences in each enhancer that
match published core Fkh-binding sites
(Lehmann and Korge, 1996;
Mach et al., 1996;
Lehmann et al., 1997). Fkh
bound strongly to site `d' in the SG1 972 enhancer, as determined by
electrophoretic mobility shift assays (EMSA)
(Fig. 2A, part a). This
consensus-binding site maps 100 bp upstream of the ATG start codon. Weak
and potentially non-specific binding was also observed with site `b', which
maps 230 nucleotides upstream (Fig.
2A, part b, left gel; increased amounts of unlabeled site `b' DNA
did not decrease binding to labeled site `d'). Double-stranded oligos
corresponding to the two remaining consensus Fkh-binding sequences in the
SG1 972 enhancer were not bound by Fkh (sites `a' and `c') and did
not compete for binding to site `d' by EMSA (data not shown). Fkh bound to
three out of the four consensus binding sequences in the SG2 885
enhancer. DNA fragments corresponding to sites `f' and `h' were bound strongly
by Fkh (Fig. 3A, parts a,b,
left gels), whereas site `g' showed moderate binding and did not compete well
with the strong binding site `h' (data not shown). The in vitro Fkh binding
sites in SG2 map between 120 and 170 nucleotides upstream of the
start codon.
To test if regulation of SG1 and SG2 is through the in vitro Fkh-binding sites, we mutated the SG1 and SG2 Fkh-binding sites in each reporter gene construct. Double-stranded DNA fragments corresponding to these mutations did not compete for in vitro binding of Fkh by EMSA (Fig. 2A, part a; Fig. 3A, parts a,b, right gels; data not shown). Surprisingly, expression of ßgal from SG1 972 fkh-lacZ (sites `b' and `d' mutated) showed little change in salivary gland expression (Fig. 2E-G). Expression of ßgal from SG2 885 fkh-lacZ (sites `f', `g' and `h' mutated) was distinct from wild-type SG2 885-lacZ; early expression was reduced and later expression was variable (Fig. 3E-G). These results indicate that expression of SG1 is unlikely to be controlled directly by Fkh and that expression of SG2 is only partially directly dependent on Fkh.
Fkh is required for expression of Sage
The above experiments suggest that although Fkh is required for
SG1 and SG2 expression, its role is likely to be at least
partly indirect and through other Fkh-dependent transcription factors.
sage, which encodes a bHLH transcription factor expressed in only the
salivary gland, is an attractive candidate for contributing to SG1
and SG2 activation (Fig.
4A) (Moore et al.,
2000; Chandrasekaran and
Beckendorf, 2003). sage is first detected during
embryonic stage 10, about the same time as fkh expression is first
observed, and continues to be expressed in the salivary gland through the
remainder of embryogenesis (Fig.
4A) and throughout larval life
(Li and White, 2003). In
fkh mutants, initial expression of sage was unaltered;
however, sage expression disappeared during embryonic stage 12 and
was undetectable by embryonic stage 13
(Fig. 4B). Thus, Fkh is
required to maintain but not initiate sage expression.
|
230 nucleotides upstream of the start codon and
matches 16/16 nucleotides of one of the two strongest in vitro Fkh binding
sites in the SG2 885 enhancer (site `h';
Fig. 3). To test if Fkh works
through this site to maintain sage expression, we established
transgenic fly lines carrying the sage reporter gene construct in
which the Fkh site was mutated. ßgal expression from all four Sage
1.2 fkh-lacZ lines was reduced compared with expression from lines
carrying the wild-type version of the construct
(Fig. 5F,G). Thus, the
Fkh-binding site in the sage enhancer is required for wild-type
levels of expression, but the relatively high level of expression that
persists suggests that other factors must also contribute. The wild-type sage reporter gene construct also contains five consensus bHLH-binding sites (CANNTG; Fig. 5A). To test whether sage auto-regulates through these sites, we created transgenic lines carrying mutated versions of the reporter construct in which either only the bHLH consensus sites were mutated (Sage 1.2 bHLH-lacZ) or both the bHLH sites and Fkh site were mutated (Sage 1.2 fkh bHLH-lacZ). Sage 1.2 bHLH-lacZ had reduced ßgal expression compared with levels from the wild-type construct (compare Fig. 5H,I with Fig. 5B,C). Sage 1.2 fkh bHLH-lacZ lines expressed ßgal to levels seen with the wild-type construct in fkh mutants (compare Fig. 5J,K with Fig. 5D,E). These findings suggest that both Fkh and a bHLH protein directly activate the robust levels of sage expression observed in the wild-type salivary gland. Although we cannot definitively identity the bHLH protein involved, we favor the idea that Sage autoactivates via the bHLH binding sites.
Sage and Fkh control high level SG2 expression
To determine whether Sage activates expression of SG1 or
SG2, we would ideally examine their expression in sage
loss-of-function mutants, but such mutations have not been reported and the
deficiency that removes sage also removes neuralized, a
neurogenic gene required to form non-neuronal ectodermal derivatives in the
ventral region of the embryo, including salivary glands
(Hartenstein et al., 1992).
Therefore, we asked if expression of Sage in new places induces ectopic
expression of either SG1 or SG2, using a ubiquitously
expressed tubulin-GAL4 to drive expression of a wild-type
sage cDNA (tub-GAL4:UAS-sage). In these embryos, both
SG1 and SG2 mRNAs were expressed at ectopic sites. In
addition to its normal salivary gland expression, SG1 mRNA was
detected in paired ectodermal stripes within each segment, as well as in the
foregut and hindgut during stage 11 (Fig.
6A, part a). At later stages, SG1 mRNA was detected in
the foregut, hindgut and Malpighian tubules. Ectopic SG2 mRNA was
detected in regions of the foregut and hindgut, and in the Malpighian tubules
(Fig. 6Ab). Thus, Sage can
activate expression of SG1 and SG2 in new places, making it
a likely regulator for both genes. Importantly, the ectopic domains of
SG1 and SG2 expression were limited to cells that also
express Fkh (Fig. 6A, part c),
suggesting that Fkh may also regulate an essential co-factor for Sage,
potentially a bHLH partner protein.
|
ßgal expression from the construct containing the SG2 enhancers with mutations in the consensus bHLH-binding sites (SG2 885 bHLH-lacZ) was somewhat reduced and variable at later stages compared with expression of the wild-type construct (SG2 885-lacZ; Fig. 6Ca). ßgal expression was nearly absent with the construct in which both the Fkh and consensus bHLH sites were mutated (SG2 885 fkh bHLH-lacZ; Fig. 6C, part b). These findings indicate that SG2 expression is controlled directly by a combination of Fkh and a bHLH protein, most probably Sage.
SG1 and SG2 are required for maintaining the salivary gland lumena
To determine which aspects of Fkh (and Sage) function SG1 and/or
SG2 mediate, we examined Crumbs (Crb) staining in the salivary glands
of Df(3R)Exel6216 embryos; Crb spans the apical membranes of
epithelial cells, with high levels in a domain just apical to the adherens
junctions, allowing examination of lumen morphology. Df(3R)Exel6216
removes most of the genes in the PH4 99F complex, including
SG1 and SG2 (Fig.
7A). In embryos stage 15 and older, defects in the apical surfaces
of the secretory cells were consistently observed; specifically, the lumena
were irregular with regions where intense Crb staining appeared to span across
the lumen (Fig. 7B). Confocal
imaging of salivary glands stained with different combinations of nuclear
(CrebA), apical (Crb, ßHSpectrin), subapical (Catenin)
and basolateral markers (Spectrin) revealed gross irregularities in the
salivary gland lumena in 100% of deficiency embryos embryonic stage 15 and
older (Fig. 7C). The lumena had
regions of expansion and constriction, as well as regions where the tubes
appeared closed [Fig. 7C
(asterisks); data not shown]. Confocal sections revealed that the small lumena
were often surrounded by Crb and ßHSpectrin staining
(Fig. 7C, middle right panels;
data not shown), again suggesting that the tubes are occluded.
Three-dimensional projections of salivary glands stained for CrebA and Crb
revealed lumena of uniform diameter in late stage wild-type salivary glands
that were never observed in the deficiency embryos; instead, the lumena from
deficiency embryos were always irregular, with regions of both dilation and
constriction (Fig. 7D). Cell
polarity and other aspects of gland morphology were not significantly altered,
although the mutant glands displayed some level of curvature and shape
irregularities, probably owing to the changes in cell contact at the apical
surfaces.
|
To gain insight into the Df(3R)Exel6216 phenotype, salivary glands were imaged by transmission electron microscopy (TEM). TEMs of wild-type stage 16/17 salivary glands showed relatively uniformly sized lumena with apparent narrowing only in the regions where the salivary glands normally curve (Fig. 8A-A'''). By contrast, TEMs of salivary glands from the deficiency embryos revealed gross lumenal irregularities, with small lumena surrounded by large regions where the tubes appeared to be closed (Fig. 8B-B''',C-C'). Given the irregular lumenal morphologies observed in the deficiency embryos with confocal microscopy, it is likely that more lumena are present in these glands than is suggested by the TEMs; it is simply the irregularity of the lumen that limits the amount captured in a single thin section. High-magnification TEM images revealed structures characteristic of adherens junctions (AJs) adjacent to the small lumena (arrowheads in Fig. 8C'). These junctions could be newly formed complexes created when cells that were originally separated by the lumen came into direct contact.
|
|
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
).
Nonetheless, our studies reveal notable differences in the roles of the two
genes in organ formation. PHA-4 is proposed to directly regulate expression of
all pharynx-specific genes (Gaudet and
Mango, 2002), whereas Fkh is required for the expression of only
about one-third of the salivary gland-specific genes tested so far (>200
genes; E. Grevengoed and D.J.A., unpublished). Moreover, many downstream genes
are indirect targets of Fkh. For example, although Fkh is required for
high-level expression of 34 secretory pathway component genes, its role in
their activation is largely indirect through maintenance of CrebA, which is
more directly involved in the expression of these genes
(Abrams and Andrew, 2005). As
observed with the secretory pathway genes, more than half of the Fkh targets
require Fkh only for maintenance, not initiation (E. Grevengoed and D.J.A.,
unpublished), suggesting that regulation is mediated by Fkh-dependent
downstream transcription factors. Even with SG1 and SG2,
which absolutely require Fkh for all stages of expression, only SG2
appears to be regulated directly by Fkh. Fkh regulation of SG1
appears to be through an intermediate, currently unidentified transcription
factor, even though the SG1 enhancer contains two sites that bind Fkh
protein in vitro. Indeed, detailed enhancer analyses have revealed only a
small number of direct transcriptional targets of Fkh, including both
sage and CrebA, which encode transcription factor genes
that, in turn, either function downstream of or in parallel with Fkh to
regulate gene expression. Thus, although Fkh functions as a key regulator of
salivary gland development, it does so in collaboration with other early
expressed transcription factors.
|
;
Mach et al., 1996;
Lehmann et al., 1997), which
revealed a seven bp core consensus Fkh-binding site. Although we found four
such consensus sites within each enhancer, not all of the sites were bound by
Fkh protein in vitro. Three sites showed strong binding (`d', `f' and `h'),
two sites showed moderate (`g') or weak (non-specific) binding (`b') and three
sites were not bound at all (`a', `c' and `e'). Subsequently, Takiya et al.
(Takiya et al., 2003) reported
Fkh DNA binding to be strongly influenced by negative cooperativity among
neighboring bases. Our binding data are consistent with their findings. The
three strong Fkh-binding sites in the SG1 (`d') and SG2 (`f'
and `h') enhancers matched the optimal sequences for binding determined by
Takiya et al. (Takiya et al.,
2003) in all positions (Table
1). The moderate Fkh-binding site (`g') had a C in position 11,
which was shown to reduce binding affinity. The weak Fkh-binding site (`b')
had an inhibitory T9A10 dinucleotide, and the three
sites that did not bind Fkh at all (`a', `c' and `e') each had an unfavorable
A7T8 dinucleotide as well as nucleotides unfavorable for
binding at positions 9 and/or 11. Our demonstration that the Fkh-binding sites
within both the SG2 and sage enhancers are required for
their full level salivary gland expression indicates a direct correlation
between in vitro studies and site occupancy in vivo; sites that function in
vivo are bound by the Fkh protein in vitro. Our studies of SG1
regulation, however, indicate that not all sites bound by Fkh protein in vitro
are necessary in vivo as SG1 reporter gene expression was unaltered
when the Fkh sites were disrupted.
|
|
;
Araujo et al., 2005;
Devine et al., 2005;
Tonning et al., 2005).
Jazwinska et al. (Jazwinska et al.,
2003) showed that two apical proteins, Piopio (Pio) and Dumpy
(Dp), are required during secondary branch formation. In this process, cells
that are arrayed side-by-side in a multicellular tube rearrange to an
end-to-end configuration to form unicellular tubes, while maintaining tube
integrity with uniform lumenal space. In pio or dp mutants,
tracheal cells detach from the main tracheal artery just as they complete
their rearrangements to form unicellular tubes. The authors suggest that this
occurs because the formation of the autocellular junctions of the unicellular
tubes continues to completion instead of stopping at the point where the cells
contact their proximal neighbors. The authors further suggest that Pio and Dp
contribute to an apical ECM that prevents reduction in lumen diameter as the
secondary branches form. A role for an apical ECM in maintaining tube diameter
has also been discovered in the multicellular tubes of the dorsal trunk
(Araujo et al., 2005;
Devine et al., 2005;
Tonning et al., 2005). Genetic
or pharmacological disruptions in chitin synthesis lead to regions of tube
constriction and dilation. Tonning et al.
(Tonning et al., 2005) showed
the existence of a transient chitin network in the tracheal lumen that ensures
that as the different segments of the dorsal trunk fuse to form a continuous
tube, uniform tube diameter is maintained.
Our studies demonstrate a correlation between apical ECM volume/morphology
and lumen size uniformity, even in tubes not undergoing extensive cell
rearrangements. The phenotypes of SG1/SG2-deficient salivary glands
suggest that the apical ECM has both a barrier function that prevents cells
from contacting one another and closing the tube, as well as a scaffolding
function that prevents lumenal dilation. Similar defects were observed with
mutations in pasilla (ps), which encodes a splicing factor
homologous to the mammalian proteins Nova1 and Nova2
(Seshaiah et al., 2001). At
the TEM level, ps mutants exhibit a decrease in secreted lumenal
content and a reduction in the number and size of apical secretory granules.
Although, as a splicing factor, Pasilla must be acting indirectly to affect
secretion levels, its phenotype demonstrates a direct correlation between
secretory volume and lumen size uniformity, a correlation supported by the
defects in SG1/SG2-deficient glands. As SG1 and SG2
encode enzymes that could modify proteins in apical secretions, their role in
this process is likely to be more direct.
The apical matrix of wild-type glands forms a fibrillar network structure
that is not apparent in salivary glands of embryos missing SG1 and
SG2. This phenotype suggests that protein modification by the SG1 and
SG2 prolyl-4-hydroxylases (PH4s) alters the character of secreted apical
proteins to allow them to form fibrillar structures that maintain an expanded
network of ECM. A role for prolyl hydroxylation in the formation of fibrillar
collagen has been known for decades (for a review, see
Myllyharju and Kivirikko,
2004). Although canonical collagens are not expressed in the
Drosophila salivary gland, a large number of uncharacterized genes
encoding secreted proteins that contain the Pro-X-Gly repeats exist, which
could be substrates for SG1/SG2 prolyl hydroxylation. Collagens are the major
protein components of the ECM, where they serve key structural roles as
exemplified by mutations in the human genes that lead to fragile bones, bone
and joint deformities, as well as fragile skin and blood vessels (for a
review, see Myllyharju and Kivirikko,
2004). A `structural' role for a collagen-related protein(s) in
the apical matrix of salivary glands is consistent with our observations.
Interestingly, formation of collagen fibrils occurs post-secretion, where
enzymes outside the cell remove the propeptides from procollagen to allow
fibrillar collagen formation. Similarly, the fibrillar nature of the lumenal
secretions of the wild-type Drosophila salivary gland is not visible
in the subapical secretory vesicles, suggesting that the fibrillar structures
found in the apical matrix also form post-secretion. We propose that the
denser apical matrix with reduced volume is the basis for the defects observed
in SG1/SG2-deficient salivary glands. In areas where there is little
to no apical content, the opposing sides of the tubes meet to either close or
form very small lumena lined with small apical surfaces and closely arrayed
adherens junctions. The similarity of the SG1/SG2 deficiency
phenotypes to those seen with mutations affecting the Drosophila
trachea suggests the potential for shared mechanisms for maintaining lumen
size uniformity in epithelial tubes.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Abrams, E. W. and Andrew, D. J. (2002). Prolyl
4-hydroxylase -related proteins in Drosophila melanogaster:
tissue-specific embryonic expression of the 99F8-9 cluster. Mech.
Dev. 112,165
-171.[CrossRef][Medline]
Abrams, E. W. and Andrew, D. J. (2005). CrebA
regulates secretory activity in the Drosophila salivary gland and
epidermis. Development
132,2743
-2758.
Abrams, E. W., Vining, M. S. and Andrew, D. J.
(2003). Constructing an organ: the Drosophila salivary
gland as a model for tube formation. Trends Cell Biol.
13,247
-254.[CrossRef][Medline]
Andrew, D. J., Horner, M. A., Petitt, M. G., Smolik, S. M. and
Scott, M. P. (1994). Setting limits on homeotic gene
function: restraint of Sex combs reduced activity by teashirt and
other homeotic genes. EMBO J.
13,1132
-1144.[Medline]
Andrew, D. J., Baig, A., Bhanot, P., Smolik, S. M. and
Henderson, K. D. (1997). The Drosophila dCREB-A gene
is required for dorsal/ventral patterning of the larval cuticle.
Development 124,181
-193.[Abstract]
Andrew, D. J., Henderson, K. D. and Seshaiah, P.
(2000). Salivary gland development in Drosophila
melanogaster. Mech. Dev. 92,5
-17.[CrossRef][Medline]
Araujo, S. J., Aslam, H., Tear, G. and Casanova, J.
(2005). Mummy/cystic encodes an enzyme required for chitin and
glycan synthesis, involved in trachea, embryonic cuticle and CNS
development-Analysis of its role in Drosophila tracheal morphogenesis.
Dev. Biol. 288,179
-193.[CrossRef][Medline]
Bradley, P. L., Haberman, A. S. and Andrew, D. J.
(2001). Organ formation in Drosophila: specification and
morphogenesis of the salivary gland. BioEssays
23,901
-911.[CrossRef][Medline]
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.[Abstract]
Chandrasekaran, V. and Beckendorf, S. K.
(2003). senseless is necessary for the survival of
embryonic salivary glands in Drosophila. Development
130,4719
-4728.
Chartier, A., Zaffran, S., Astier, M., Semeriva, M. and
Gratecos, D. (2002). Pericardin, a Drosophila type
IV collagen-like protein is involved in the morphogenesis and maintenance of
the heart epithelium during dorsal ectoderm closure.
Development 129,3241
-3253.
Devine, W. P., Lubarsky, B., Shaw, K., Luschnig, S., Messina, L.
and Krasnow, M. A. (2005). Requirements for chitin
biosynthesis in epithelial tube morphogenesis. Proc. Natl. Acad.
Sci. USA 102,17014
-17019.
Gaudet, J. and Mango, S. E. (2002). Regulation
of organogenesis by the Caenorhabditis elegans FoxA protein PHA-4.
Science 295,821
-825.
Haberman, A. S., Isaac, D. D. and Andrew, D. J.
(2003). Specification of cell fates within the salivary gland
primordium. Dev. Biol.
258,443
-453.[CrossRef][Medline]
Hartenstein, A. Y., Rugendorff, A., Tepass, U. and Hartenstein,
V. (1992). The function of the neurogenic genes during
epithelial development in the Drosophila embryo.
Development 116,1203
-1220.[Abstract]
Henderson, K. D. and Andrew, D. J. (2000).
Regulation and function of Scr, exd, and hth in the
Drosophila salivary gland. Dev. Biol.
217,362
-374.[CrossRef][Medline]
Horner, M. A., Quintin, X., Domeier, M. E., Kimble, J.,
Labouesse, M. and Mango, S. E. (1998). pha-4, an
HNF-3 homolog, specifies pharyngeal organ identity in Caenorhabditis
elegans. Genes Dev. 12,1947
-1952.
Jazwinska, A., Ribiero, C. and Affolter, M.
(2003). Epithelial tubule morphogenesis during
Drosophila tracheal development requires Piopio, a lumenal ZP
protein. Nat. Cell Biol.
5, 895-901.[CrossRef][Medline]
Kalb, J. M., Lau, K. K., Goszczynski, B., Fukushige, T., Moons,
D., Okkema, P. G. and McGhee, J. D. (1998). pha-4 is
Ce-fkh-1, a fork head/HNF-3alpha,beta,gamma homolog that functions in
organogenesis of the C. elegans pharynx.
Development 125,2171
-2180.[Abstract]
Kivirikko, K. I. and Pihlajaniemi, T. (1998).
Collagen hydroxylases and the protein disulfide isomerase subunit of prolyl
4-hydroxylases. Adv. Enzymol. Relat. Areas Mol. Biol.
72,325
-398.[Medline]
Knibiehler, B., Mirre, C. and Le Parco, Y.
(1990). Collagen type IV of Drosophila is stockpiled in
the growing oocyte snd differentially located during early stages of
embryogenesis. Cell Differ. Dev.
30,147
-157.[CrossRef][Medline]
Kuo, Y. M., Jones, N., Zhou, B., Panzer, S., Larson, V. and
Beckendorf, S. K. (1996). Salivary duct determination in
Drosophila: roles of the EGF receptor signalling pathway and the transcription
factors fork head and trachealess. Development
122,1909
-1917.[Abstract]
Le Parco, Y., Knibiehler, B., Cecchini, J. P. and Mirre, C.
(1986). Stage and tissue-specific expression of a collagen gene
during Drosophila melanogaster development. Exp. Cell
Res. 163,405
-412.[CrossRef][Medline]
Lee, T. and Luo, L. (1999). Mosaic analysis
with a repressible cell marker for studies of gene funciton in neuronal
morphogenesis. Neuron
22,451
-461.[CrossRef][Medline]
Lehmann, M. and Korge, G. (1996). The fork
head product directly specifies the tissue-specific hormone
responsiveness of the Drosophila Sgs-4 gene. EMBO
J. 15,4825
-4834.[Medline]
Lehmann, M., Wattler, F. and Korge, G. (1997).
Two new regulatory elements controlling the Drosophila Sgs-3 gene are
potential ecdysone receptor and fork head binding sites. Mech.
Dev. 62,15
-27.[CrossRef][Medline]
Lehmann, R. and Tautz, D. (1994). In situ
hybridization to RNA. Methods Cell Biol.
44,575
-598.[Medline]
Li, T. R. and White, K. P. (2003).
Tissue-specific gene expression and ecdysone-regulated genomic networks in
Drosophila. Dev. Cell 5,59
-72.[CrossRef][Medline]
Mach, V., Ohno, K., Kokubo, H. and Suzuki, Y.
(1996). The Drosophila Fork head factor directly
controls larval salivary gland-specific expression of the glue protein gene
Sgs3. Nucleic Acids Res.
24,2387
-2394.
Moore, A. W., Barbel, S., Jan, L. Y. and Jan, Y. N.
(2000). A genome-wide survey of basic helix-loop-helix factors in
Drosophila. Proc. Natl. Acad. Sci. USA
97,10436
-10441.
Myat, M. M. and Andrew, D. J. (2000). Fork head
prevents apoptosis and promotes cell shape change during formation of the
Drosophila salivary glands. Development
127,4217
-4226.[Abstract]
Myllyharju, J. and Kivirikko, K. I. (2004).
Collagens, modifying enzymes and their mutations in humans, flies and worms.
Trends Genet. 20,33
-43.[CrossRef][Medline]
Oda, H., Uemura, T., Harada, Y., Iwai, Y. and Takeichi, M.
(1994). A Drosophila homolog of cadherin associated with
armadillo and essential for embryonic cell-cell adhesion. Dev.
Biol. 165,716
-726.[CrossRef][Medline]
Panzer, S., Weigel, D. and Beckendorf, S. K.
(1992). Organogenesis in Drosophila melanogaster: embryonic
salivary gland determination is controlled by homeotic and dorsoventral
patterning genes. Development
114, 49-57.[Abstract]
Parks, A. L., Cook, K. R., Belvin, M., Dompe, N. A., Fawcett,
R., Huppert, K., Tan, L. R., Winter, C. G., Bogart, K. P., Deal, J. E. et
al. (2004). Systematic generation of high-resolution deletion
coverage of the Drosophila melanogaster genome. Nat.
Genet. 36,288
-292.[CrossRef][Medline]
Reuter, R., Panganiban, E. E., Hoffman, F. M. and Scott, M.
P. (1990). Homeotic genes regulate the spatial expression of
putative growth factors in the visceral mesoderm of Drosophila embryos.
Development 110,1031
-1040.
Seshaiah, P., Miller, B., Myat, M. M. and Andrew, D. J.
(2001). pasilla, the Drosophila homologue of the human
Nova-1 and Nova-2 proteins, is required for normal secretion in the salivary
gland. Dev. Biol. 239,309
-322.[CrossRef][Medline]
Spradling, A. C. and Rubin, G. M. (1982).
Transpositon of cloned P elements into Drosophila; germ line chromosomes.
Science 218,341
-347.
Takiya, S., Gazi, and Mach, V. (2003). The DNA
binding of insect Fork head factors is strongly influenced by the negative
cooperation of neighboring bases. Insect Biochem. Mol.
Biol. 33,1145
-1154.[CrossRef][Medline]
Thomas, G. H. and Kiehart, D. P. (1994). Beta
hevy-spectrin has a restricted tissue and subcellular distribution during
Drosophila embryogenesis. Development
124,2039
-2050.
Thummel, C. S., Boulet, A. M. and Lipshitz, H. D.
(1988). Vectors for Drosophila P-element-mediated transformation
and tissue culture transfection. Gene
74,445
-456.[CrossRef][Medline]
Tonning, A., Hemphala, J., Tang, E., Nannmark, U., Samakovlis,
C. and Uv, A. (2005). A transient lumenal chitinous matrix is
required to model epithelial tube diameter in the Drosophila trachea.
Dev. Cell 9,423
-430.[CrossRef][Medline]
Weigel, D., Bellen, H. J., Jürgens, G. and Jäckle,
H. (1989a). Primordium specific requirement of the homeotic
gene fork head in the developing gut of the Drosophila
embryo. Rouxs Arch. Dev. Biol.
198,201
-210.[CrossRef]
Weigel, D., Jurgens, G., Kuttner, F., Seifert, E. and Jackle,
H. (1989b). The homeotic gene fork head encodes a
nuclear protein and is expressed in the terminal regions of the
Drosophila embryo. Cell
57,645
-658.[CrossRef][Medline]
Yasothornsrikul, S., Davis, W. J., Cramer, G., Kimbrell, D. and
Dearolf, C. R. (1997). viking: identification and
charcterization of a second type IV collagen in Drosophila.Gene 198,17
-25.[CrossRef][Medline]
Zhou, B., Bagri, A. and Beckendorf, S. K.
(2001). Salivary gland determination in Drosophila: a
salivary-specific, fork head enhancer integrates spatial pattern and
allows fork head autoregulation. Dev. Biol.
237, 54-67.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  Q. Xia, Y. Guo, Z. Zhang, D. Li, Z. Xuan, Z. Li, F. Dai, Y. Li, D. Cheng, R. Li, et al. Complete Resequencing of 40 Genomes Reveals Domestication Events and Genes in Silkworm (Bombyx) Science, October 16, 2009; 326(5951): 433 - 436. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Maybeck and K. Roper A Targeted Gain-of-Function Screen Identifies Genes Affecting Salivary Gland Morphogenesis/Tubulogenesis in Drosophila Genetics, February 1, 2009; 181(2): 543 - 565. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Keskiaho, L. Kukkola, A. P. Page, A. D. Winter, J. Vuoristo, R. Sormunen, R. Nissi, P. Riihimaa, and J. Myllyharju Characterization of a Novel Caenorhabditis elegans Prolyl 4-Hydroxylase with a Unique Substrate Specificity and Restricted Expression in the Pharynx and Excretory Duct J. Biol. Chem., April 18, 2008; 283(16): 10679 - 10689. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Harris and S. K. Beckendorf Different Wnt signals act through the Frizzled and RYK receptors during Drosophila salivary gland migration Development, June 1, 2007; 134(11): 2017 - 2025. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Cao, Y. Liu, and M. Lehmann Fork head controls the timing and tissue selectivity of steroid-induced developmental cell death J. Cell Biol., March 12, 2007; 176(6): 843 - 852. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
