Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA
* Author for correspondence (e-mail: miketiemeyer{at}eurofinsus.com)
Accepted 16 December 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Toll-like receptor, Glycosylation, HRP epitope, N-linked oligosaccharide, Neuronal differentiation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies raised against the plant glycoprotein, Horseradish Peroxidase
(HRP), crossreact with an N-linked oligosaccharide epitope that is distributed
throughout the Drosophila melanogaster nervous system and is also
expressed in a small, well characterized subset of non-neural tissues
(Jan and Jan, 1982;
Snow et al., 1987
). Two
mutations abolish expression of the HRP epitope. In the first, designated
nac, the epitope is absent in the larval, pupal and adult nervous
system (Katz et al., 1988
).
The molecular nature of the nac mutation is unknown, but affected
adults exhibit sensory afferent defasciculation and behavioral phenotypes
(Whitlock, 1993
;
Phillis et al., 1993
). The
second mutation that abolishes HRP-epitope expression is carried on the TM3
balancer chromosome, an extensively rearranged form of the third chromosome
(Snow et al., 1987
). TM3
homozygotes do not express the HRP epitope in the embryonic nervous system but
do produce the glycan in the expected non-neural tissues. Therefore, it is
likely that the structural genes necessary for synthesis of the HRP epitope
are intact and that the TM3 mutation alters a gene that regulates
tissue-specific glycosylation.
We report characterization of the TM3 locus that abolishes HRP-epitope
expression. The affected gene, which we named `tollo' encodes a
member of the family of cell surface receptors with homology to the Toll
protein (Toll-like receptors, TLRs). Genome sequence characterization
re-identified the tollo locus, resulting in its designation as
`toll-8' (Tauszig et al.,
2000). The founding member of the TLR family (Toll) was originally
identified as a component of the signaling pathway that induces dorsal-ventral
polarity in the Drosophila embryo
(Anderson et al., 1985
).
Subsequently, TLRs have also been shown to participate in innate immune
responses in Drosophila and other organisms by transducing pattern
recognition signals (Hoffman et al.,
1999
; Medzhitov et al.,
1997
; Williams et al.,
1997
; Ip and Levine,
1994
). We now add the induction of tissue-specific glycosylation
to the list of TLR functions.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fly stocks and deletion screens
A non-complementation screen using the third chromosome deletion set
(Bloomington Stock Center) was performed to determine which region of the TM3
balancer chromosome is responsible for loss of the HRP epitope. For deletion
stocks maintained over the TM3 balancer, embryos were collected and stained
for loss of the HRP epitope. For stocks not already over the TM3 balancer,
progeny were collected from a cross of the deletion stock to a TM3 stock in
which the balancer carried an embryonically expressed lacZ marker
(D, ry/TM3-DZ, P{ry+t7.2=H22.7}, Bloomington Stock
Center). The lacZ activity, detectable in maxillary segments from
stage 12-17, allowed the HRP-status of blue embryos to be interpreted as
complementation/non-complementation of the TM3 mutation.
Once a region of interest was identified (by the fzM21 interval 70D2/3-71E4/5), additional deletions were obtained to refine the location of the affected TM3 locus. Deletion lines and their breakpoints from sources other than the Bloomington Stock Center were as follows: D5rv14 (70D1/2-71C1/2; A. Carpenter, University of Cambridge), Brd15 (71A1/2-71C1/2; J. Posakony, University of California at San Diego) and fzD21 (70D2-70E8, Umeå Stock Center). Other deletions were obtained from the Bloomington Stock Center: Df(3L)BK10 (71C3 - 71E5), D5rv5 (70C3/4 - 70F5/71A1), fzGS1a (70D1 - 70E7) and fzGF3b (70C1/2 - 70D4/5). The Gal4 driver stocks, rhomboid-Gal4, ELAV-Gal4 and hsp70-Gal4 were obtained from Tian Xu and a second chromosome UAS-lacZ line was obtained from Haig Keshishian (both at Yale University).
Immunohistochemistry, lacZ activity staining and in situ
hybridization
Embryo collections were dechorionated, fixed, devitellinized, stained with
antibodies and staged according to standard methods
(Patel, 1994;
Campos-Ortega and Hartenstein,
1985
). Primary antibody dilutions were 1:500 for rabbit anti-HRP,
1:3 for BP102, 1:5 for 22C10 and 1:10 for 1D4. lacZ activity was detected in
embryos as previously described
(Klämbt et al., 1991
).
The distribution of tollo mRNA in embryos was visualized by in situ
hybridization using single-stranded DNA probes labeled with digoxigenin
(Tautz and Pfeifle, 1989
). For
co-localization of tollo mRNA and mAb 22C10 staining, in situ
hybridization was performed first, using digoxigenin-11-UTP-labeled RNA probes
prepared by in vitro transcription
(Kopczynski et al., 1996
). For
RNA and DNA probes, a 2.4 kb EcoR1 genomic fragment (tollo nucleotide
2796 to 5230) served as template. Embryos were routinely examined with sense
and antisense probes prepared from plasmid templates bearing insert in
opposite orientations.
Reverse northern analysis, sequence assembly and genomic
characterization
Poly-A+ RNA was isolated from OreR or
Brd15/TM3 embryonic total RNA by hybridization to
biotinylated poly-dT oligonucleotide followed by capture and release from
streptavidin magnetic beads as recommended by the manufacturer
(Boehringer/Roche). To generate probe for Reverse Northern analysis,
poly-A+ mRNA was dephosphorylated with calf intestinal phosphatase
and then end-labeled with -32P-ATP by T4 Polynucleotide
Kinase (Sambrook et al.,
1989
). Drosophila genomic DNA in P1 phage was digested with
restriction enzymes, electrophoresed, transferred to nylon and probed with
end-labeled mRNA prepared from OreR or Brd15/TM3
embryos.
A 2.4 kb EcoRI fragment that demonstrated differential hybridization was isolated from P1 phage DS06206, subcloned into pBluescript (Stratagene) and sequenced in both directions by multiple rounds of overlapping sequence acquisition with the dideoxy chain termination method (W. M. Keck, Biotechnology Resource Center at Yale University). The open-reading frame (ORF) identified in the EcoRI fragment was extended in the 5' direction by overlapping sequencing reactions that used DNA isolated from P1 phage DS06206 as template. The resulting genomic DNA sequence, containing a 4038 nucleotide ORF, 17 nucleotides of 5'-, and 1189 nucleotides of 3'-UTR, was submitted to GenBank (Accession Number, AF204158).
Sequence was extended in the 3' direction by 3'-RACE (Clontech
Marathon) with poly-A+ RNA isolated from OreR or
Brd15/TM3 embryos and nested primer sets
(Frohman, 1993). Distinct
bands of 1.6 kb from OreR and 1.2 kb from Brd15/TM3 were
obtained. Primer design incorporated restriction sites (5' PstI
and 3' XhoI) for ease of subcloning. The final 3'-RACE
reactions were digested with PstI and XhoI, yielding
fragments (1.1 kb from OreR and 0.9 kb from Brd15/TM3)
that were subcloned into pBluescript and sequenced. Promoter components were
identified by the NNPP2.1 prediction tool (Berkeley Drosophila Genome
Project). Other sequence analysis used BLAST programs available through the
National Center for Biotechnology Information
(Altschul et al., 1997
).
Northern analysis used poly-A+ RNA isolated from overnight embryo
collections and genomic Southern analysis used genomic DNA prepared from
adults. Both blotting procedures were performed by standard methods using
random-primed 32P-labeled DNA probes
(Sambrook et al., 1989
).
Generation of rescue constructs and transformant lines
Plasmid bearing a full-length tollo insert was constructed with
DNA from two overlapping Drosophila genomic clones in P1 phage. The
2.4 kb EcoRI fragment isolated from P1 clone DS06206, containing
3' tollo sequence, was ligated into the EcoRI site of
pBluescript I (KS)+. The resulting plasmid was digested with SalI
(cuts in the 5' polylinker) and MluI (cuts in tollo
3'-UTR) to accept a 7.5 kb SalI/MluI fragment prepared
from P1 clone DS05329. In addition to a complete tollo ORF, the
resulting plasmid (designated pGtollo) contains 2.5 kb of 5'
and 1.2 kb of 3' genomic sequence.
To generate a rescue construct in which tollo coding sequence was
placed under control of UAS elements, a NotI site was introduced into
the full-length tollo construct (pGtollo) by PCR such that
the enzyme cut 11 nucleotides upstream from the first ATG codon. NotI
digest of the resulting construct, designated pGtolloN-11,
released a 5.2 kb fragment that was ligated into NotI-linearized
pUAST transformation vector to produce pUASTtollo
(Brand et al., 1994).
To generate a transformation construct in which Gal4 expression was
controlled by tollo 5' genomic sequence, a NotI site
was introduced into cloned genomic DNA by PCR (pGtollo template) such
that the enzyme cut seven nucleotides upstream from the first ATG codon. The
amplified fragment was subcloned into pCR2.1 and recovered as a 2.5 kb
fragment with a 5' blunted SalI end and a 3'
NotI end. After ligation into the pCaSpeR3 transformation vector
(previously digested with StuI/NotI), Gal4 coding sequence
was added as a NotI fragment, prepared from pGaTN, to yield a
construct designated ptolloGal4
(Brand and Perrimon,
1993).
Transformant lines were produced by injection of rescue constructs into
w1118; Sb,2-3/TM6b flies by standard
procedures. Transformation with the pUASTtollo or ptolloGal4
vector yielded insertions on both the X and second chromosomes that were then
crossed into the Brd15/TM3-lacZ background.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Of the relevant genotypes, only Brd15 homozygotes display gross morphologic aberrations (Fig. 1D-G). Brd15/Brd15 embryos develop normally until stage 14 when defects in the formation of anterior terminal structures become apparent. In particular, retraction of the clypeolabrum is stalled in Brd15 homozygotes, causing the supraesophageal ganglia (embryonic brain lobes) to appear exteriorized. The head involution defect provides an unambiguous, reliable diagnostic for the Brd15 homozygous genotype (Fig. 1G, arrowhead). Examination of neural tissue integrity by monoclonal antibody staining demonstrates that longitudinal and commissural bundles are present in the central nervous system (mAb BP102), appropriate cell numbers and approximate cellular relations are preserved in the peripheral nervous system (mAb 22C10), and efferent motor pathways develop normally (mAb 1D4) in Brd15 homozygotes. Thus, neural differentiation and axon extension, to the extent that they are revealed by these mAb markers, are unaffected by Brd15.
Reverse-Northern analysis identifies a message that is differentially
expressed in wild-type and Brd15/TM3 embryos
The proximal breakpoint of the Brd15 deletion (71C1/2)
overlaps a TM3 rearrangement breakpoint at 71C
(Lindsley and Zimm, 1992).
Therefore, P1 phage clones that map to the 71C1/2 interval were obtained and
probed with 32P-end-labeled mRNA prepared from embryo collections
from OreR or Brd15/TM3 stocks. The P1 phage clone
designated DS06206 contains a 2.4 kb EcoRI fragment and a 1.1 kb
BamHI/XbaI fragment that are both transcribed in OreR but
not detected in Brd15/TM3 embryos
(Fig. 3A-C). Subsequent
sequence analysis placed the 1.1 kb fragment within the 2.4 kb fragment. Probe
prepared from the 2.4 kb EcoRI fragment was used to probe northern
blots of poly-A+ RNA isolated from OreR or
Brd15/TM3 embryos. A 6.5-7.0 kb band was identified in the
OreR preparation that was not detected in Brd15/TM3
poly-A+ RNA (Fig.
3D). Genomic Southern analysis demonstrates multiple restriction
fragment length polymorphisms in the Brd15/TM3 genotype.
Probe prepared from the 2.4 kb EcoRI fragment hybridizes to a 1.5 kb
HindIII fragment in OreR that is shifted to approximately 6 kb in
Brd15/TM3. In turn, probe prepared from the 1.5 kb
HindIII fragment identifies the same polymorphism as well as 876 bp
HindIII/Xba1 and 280 bp PstI fragments that also
differentiate the two genotypes (Fig.
3E-G).
|
|
A total of 6735 nucleotides were sequenced, extending from 17 bp upstream
of the ORF to 2677 bp beyond the first in-frame stop codon, and found to be
co-linear with genomic sequence in GenBank Accession Number AE003531 and with
Drosophila cDNA sequence LD 33590
(Adams et al., 2000;
Rubin et al., 2000
). The
sequence predicts that the HindIII, XbaI and PstI
polymorphisms observed in Brd15/TM3 lie in the 3'
UTR of the gene (Fig. 3E-G). To
more precisely define the polymorphism, 3'-RACE was performed on
poly-A+ RNA isolated from OreR and Brd15/TM3
embryos. The fragment amplified from Brd15/TM3 embryos
yielded 969 nucleotides of sequence of which the first 237 matched previously
sequenced genomic DNA. However, TM3 sequence diverged from wild-type at a
position corresponding to nucleotide 5635, 1.6 kb downstream from the first
in-frame stop codon of tollo (nucleotide 4039) and within the
3' UTR predicted by mRNA size (Fig.
3D, Fig. 4). Sequence obtained for the first 732 bases of divergence matches a
Drosophila transposable element designated `412', GenBank Accession
Number X04132 (Yuki et al.,
1986
). It was not determined whether the divergent sequence
reflects the insertion of an intact transposable element or identifies the
site of the TM3 rearrangement breakpoint previously mapped to 71C.
Tollo mRNA is expressed in non-neural ectodermal cells that
contact neural precursor cells
Expression of tollo mRNA is first detected in the cellular
blastoderm, initially as prominent bands at both ends of the embryo
(Fig. 5A). Very rapidly,
tollo mRNA appears in dorsoventral bands repeated along the entire
length of the embryo (Fig. 5B).
As germband retraction begins, late in stage 12, the bands of tollo
mRNA span the entire width of the germband
(Fig. 5C,D), placing Tollo
expression within ectodermal domains from which ventral nerve cord precursor
cells differentiate and delaminate. By the time germband retraction is
complete (stage 13), tollo mRNA expression disappears from the
ventral ectoderm that underlies the delaminated, discrete nerve cord.
Expression of the HRP epitope in the ventral nerve cord is first detected
reproducibly at stage 14, shortly after tollo mRNA decreases. Thus,
Tollo expression in ventral ectoderm coincides with a period of maximal
contact with differentiating neurons and disappears once neurons segregate
from the ectoderm to form a consolidated ventral nerve cord.
|
In the lateral ectoderm, tollo mRNA is found in distinct, segmentally repeated domains at stage 13. Together, these repeated domains form continuous anteroposterior stripes of ectodermal expression (Fig. 5E,F). Within each domain, expression is not uniform. Cells at the segment boundaries express higher levels of tollo mRNA, forming ectodermal pockets that are partly lined with Tollo-expressing cells. By early stage 15, tollo mRNA is greatly reduced in the lateral ectoderm and expressing domains are attenuated to a few cells immediately adjacent to segment boundaries. Expression of the HRP epitope in the peripheral nervous system is first detected reproducibly at stage 15, shortly after tollo mRNA expression has decreased in the lateral ectoderm.
Consistent with the determination that the TM3 chromosome has not lost the entire tollo gene (Figs 3, 4), hybridization signal was detected in TM3/TM3 embryos (Fig. 5G). Thus, RNA that contains tollo sequence is produced in TM3 embryos despite being undetectable in blotted poly-A+ mRNA preparations (Fig. 3D). Hybridization to Brd15/Brd15 embryos was not detected at any stage, indicating that the anti-sense probe is specific for tollo and does not cross-hybridize to other embryonically expressed Toll-like receptors (Fig. 5H).
The position of Tollo-expressing domains along the dorsoventral axis of the
lateral ectoderm closely approximates the site of proneural cluster formation
(Blochinger et al., 1990).
Therefore, tollo mRNA expression was localized relative to the
position of differentiating neurons in the peripheral nervous system
(Fig. 6A,B). Within the lateral
domains of Tollo expression found in each segment, maturing neurons occupy
patches that display reduced or undetectable tollo mRNA
(Fig. 6A). At stage 14, all but
the earliest neurons to differentiate (which have actively begun to migrate
away from their birthplace towards their final embryonic positions) are found
in close association with ectodermal cells that express tollo mRNA
(Fig. 6B). Thus, in the
peripheral nervous system, as in the ventral nerve cord, Tollo expression
coincides temporally with periods of neural differentiation that are
characterized by maximal contact between the ectoderm and neural precursor
cells.
|
Transgenic expression of Tollo rescues expression of the HRP
epitope
To determine whether tollo is sufficient to rescue expression of
the HRP epitope in the Brd15 homozygote, a transformation
construct was generated (pUASTtollo) that placed
tollo-coding sequence under the control of UAS elements
(Brand et al., 1994). A second
transformation construct was prepared (ptolloGal4) that placed Gal4
expression under control of 2.5 kb of Drosophila genomic DNA found
immediately upstream of the tollo initiation codon
(Brand and Perrimon, 1993
).
Tollo-Gal4 transformant lines were crossed to a UAS-lacZ
reporter line and embryo collections were stained for ß-galactosidase
activity. Both in the germband extended embryo at stage 12
(Fig. 7A,B) and in the lateral
ectoderm of the stage 13 embryo (Fig.
7C,D), lacZ activity matched the distribution of
tollo mRNA detected by in situ hybridization. Thus, the 2.5 kb of
genomic DNA incorporated into the ptolloGal4 transformation vector
contains control sequences sufficient to recapitulate normal Tollo
expression.
|
UAS-tollo and tollo-Gal4 transformant lines were separately prepared in the Brd15/TM3 background. HRP-epitope expression is absent from embryos collected from lines bearing either construct alone. However, when UAS-tollo and tollo-Gal4 lines are crossed to each other, HRP-epitope expression is rescued in embryos that lack (Brd15/TM3 and TM3/TM3 genotypes) and in embryos that possess (Brd15/Brd15) the head involution defect associated with the Brd15 deletion (Fig. 8A,D,G, see arrowhead). Thus, the head involution defect is independent of HRP-epitope expression. Tollo-Gal4/UAS-tollo rescues HRP-epitope expression in the ventral nerve cord (Fig. 8B,E,H) and in the peripheral nervous system (Fig. 8C,F,I).
|
Other Gal4 driver lines were screened for their ability to rescue the HRP epitope in UAS-tollo transformants. Neither a pan-neural driver (ELAV-Gal4) nor a mesectodermal/midline glial driver (rhomboid-Gal4) rescued oligosaccharide expression when crossed to UAS-tollo, despite their ability to drive expression in cells that make extensive contact with neuronal surfaces. Therefore, simple juxtaposition of Tollo protein and a neuron is insufficient; induction of the neuronal HRP epitope requires Tollo expression in appropriate non-neural ectodermal cells.
Heat-shock driven expression of Tollo in all cells (hsp70-Gal4/UAS-tollo) generates early embryonic lethality that precludes assessment of HRP-epitope rescue. However, in the course of these experiments, hsp70-Gal4/UAS-tollo embryos not subjected to heat shock were also collected and stained with anti-HRP antibody. Unexpectedly, unshocked embryos older than stage 15 express the HRP epitope in the salivary gland and in sensory neurons most proximal to the gland (Fig. 8J,K). Other neuronal populations were not stained, whether in the CNS or in more posterior segments of the PNS. Thus, leaky Gal4 expression in the salivary gland (verified by UAS-lacZ reporter) is sufficient to induce the HRP epitope in a tissue that does not normally express the glycan and is able to rescue the epitope in nearby sensory neurons.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The localization of Tollo expression to non-neural ectodermal cells and the rescue of neural-specific glycosylation by transgenic tollo both demonstrate that non-homologous cells modulate glycan expression in adjacent tissues. The close proximity of Tollo-expressing ectodermal cells to differentiating neurons is consistent with a molecular mechanism in which neural glycosylation is influenced by the activity of a neuronal surface receptor that directly binds ectodermal Tollo. Alternatively, the molecular activity of tollo may reside entirely within the ectodermal cell, exerting an indirect influence on neural glycosylation by propagating or attenuating instructive signals subsequently interpreted by local neurons. At present, our data cannot unambiguously distinguish whether the direct or indirect mechanism applies. However, the results of HRP-epitope rescue and Tollo misexpression studies indicate requirements that both models must satisfy.
Molecular mechanisms derived from Tollo expression, misexpression and
HRP-epitope rescue
Neuronal synthesis of the HRP-glycan is rescued in
Brd15/Brd15 embryos when Tollo is
expressed in its wild-type ectodermal pattern
(tollo-Gal4/UAS-tollo). However,
ELAV-Gal4/UAS-tollo and rho-Gal4/UAS-tollo
embryos fail to rescue the HRP epitope, despite driving misexpression in
neurons and glia that would present Tollo to neuronal surfaces at
developmental stages coincident with the normal Tollo expression pattern.
Therefore, if tollo acts directly to alter neural glycosylation,
ectodermal presentation of Tollo must be unique in comparison with expression
in other cell types that also share contact with neurons; either the Tollo
protein requires an ectodermal-specific post-translational modification for
activity or an ectodermal co-factor is necessary for appropriate presentation
to neurons. If tollo indirectly affects neural glycosylation by
generating or influencing paracrine signals sensed by differentiating neurons,
then the cellular context in which Tollo is expressed determines induction of
the HRP epitope; either the relevant paracrine influence is specifically of
ectodermal origin or a required tollo intracellular signaling pathway
is absent from neurons and glia.
The indirect mechanism is consistent with the function of other TLRs
(Belvin and Anderson, 1996;
Cantera et al., 1999
;
Halfon and Keshishian, 1998
)
and is supported by two additional observations. First, the discontinuous
distribution of tollo mRNA in the neurogenic ectoderm of the ventral
nerve cord indicates that tollo expression is limited to ectodermal
cells contacting only a subset of the total differentiating neuron pool.
Therefore, global CNS expression of the HRP epitope requires a signal
unrestricted by the need for cell-cell contact. Second, HRP-epitope expression
is rescued in PNS sensory neurons located near salivary glands that
ectopically express Tollo, consistent with the generation of a locally active
signal. Expression of the HRP epitope is not rescued in the CNS nor in more
remote parts of the PNS by hsp70-Gal4/UAS-tollo, implying
that temporal and physical barriers can limit Tollo activity.
The unexpected, ectopic expression of the HRP epitope in the secretory
epithelium of the salivary gland indicates that some developing tissues are
only one signal away from assuming an altered glycosylation phenotype. At
least within the salivary gland, this result also indicates that Tollo can act
directly or can generate an autocrine signal that autonomously modulates
glycosylation. For neural tissue, though, elaboration of the HRP epitope is a
non-autonomous neuronal behavior that requires ectodermal Tollo expression. By
analogy to Toll, soluble protein ligands (like Spätzle) are prime
candidates for the Tollo activator, but the full diversity of TLR ligands has
yet to be characterized in any organism
(Hoffman et al., 1999;
Yang et al., 1998
;
Belvin and Anderson, 1996
).
The structure of the HRP epitope predicts downstream targets of Tollo
signaling
In plants and in the Drosophila adult, HRP-epitope structure has
been demonstrated to contain an extensively trimmed high-mannose core carrying
an 3-linked Fuc residue on the internal GlcNAc of the chitobiose.
(Fabini et al., 2001
;
Kurosaka et al., 1991
). To
generate the described Drosophila HRP epitopes, high-mannose
oligosaccharides must first be trimmed to a Man3GlcNAc2
or Man2GlcNAc2 core. The core structure is then
di-fucosylated (
3 and
6), requiring the activity of two distinct
fucosyltransferases. Addition of Fuc
3 to the core requires previous
and transient addition of GlcNAc to a terminal Man, yielding a di-fucosylated
Man2/3GlcNAc2 oligosaccharide
(Fabini et al., 2001
;
Altmann et al., 1993
;
Altmann et al., 1995
).
Therefore, trimming mannosidases, two fucosyltransferases, an
N-acetylglucosaminyltransferase and a hexosaminidase constitute the minimal
set of processing activities required to generate an HRP epitope. Of these
activities, addition of the
3 Fuc imparts antibody recognition to the
oligosaccharide.
A Drosophila fucosyltransferase that adds Fuc in 3 linkage
to core GlcNAc has been characterized
(Fabini et al., 2001
).
Designated `FucTA', the enzyme exhibits in vitro acceptor specificity
appropriate for synthesis of the HRP epitope and the gene maps to 71B2, 87 kb
distal to tollo. Although this lies within the
Brd15 deletion, combining tollo-Gal4 with
UAS-tollo results in rescue of HRP epitope expression in
Brd15 homozygotes. Therefore, glycan expression is rescued
by Tollo/Toll-8 in a FucTA null background. The relevance of FucTA
activity to HRP-epitope expression in the embryonic nervous system remains to
be determined, but Drosophila requires
3 fucosyltransferase
activity and the resulting capacity to synthesize the HRP epitope in multiple
contexts. Mutants that lack the HRP epitope in larval and adult stages express
the oligosaccharide embryonically and epitope expression in embryonic
non-neural tissue is maintained in mutants that lack the embryonic neural
oligosaccharide (Snow et al.,
1987
; Katz et al.,
1988
). Thus, multiple pathways, under independent control and
active in different tissues and developmental stages, lead to synthesis of the
HRP epitope.
An oligosaccharide of unknown function reveals a new function for
Toll-like receptors
Our results suggest superficially that loss of the HRP epitope is of
relatively little consequence. However, the component of the HRP epitope
structure that imparts antibody recognition may be distinct from the
functional domain of the oligosaccharide. Therefore, mutations in genes such
as tollo, which affect specific carbohydrate expression, may not
immediately reveal oligosaccharide function. The nac mutant, which
lacks larval, pupal and adult expression of the HRP epitope, exhibits grossly
normal nervous system morphology (Katz et
al., 1988; Phillis et al.,
1993
). Highly penetrant axon defasciculation errors are present in
the nac adult but only become apparent when afferent projections
arising from discrete subsets of dye-labeled sensory neurons in the wing
margin are visualized at their entry point into the central nervous system
(Whitlock, 1993
). Until
techniques of similar resolution are applied to embryos that lack the HRP
epitope, the functional significance of loss of this tissue-specific glycan
cannot be fully evaluated.
In Drosophila, the HRP epitope is present on several neural
proteins, many of which are also expressed in non-neural tissue where they
lack the glycan (Desai et al.,
1994; Snow et al.,
1987
; Sun et al., 1995; Wang
et al., 1994
). Thus, cells determine whether or not to construct
the HRP epitope on a particular glycoprotein based on the tissue in which the
protein is expressed, rather than on a signal intrinsic to the polypeptide.
While tollo/toll-8 demonstrates that such tissue-specific glycan
expression can be achieved through the activity of a Toll-like receptor, the
correlation between Drosophila TLR expression and specific
glycosylation patterns cannot be comprehensively assessed before glycan
characterization in the Drosophila embryo is greatly expanded
(Fabini et al., 2001
;
Seppo and Tiemeyer, 2000
).
Nonetheless, distributions of other TLRs exhibit spatial and temporal overlap
with Tollo expression, raising the possibility that TLRs sculpt embryonic
glycosylation patterns through combinatorial activation of glycosylation
pathways in interacting domains of developing tissues
(Hashimoto et al., 1988
;
Eldon et al., 1994
;
Chiang and Beachy, 1994
;
Stathopolous and Levine, 2002).
TLRs mediate pattern recognition (frequently glycan-based) as part of the
innate, non-adaptive immune response in Drosophila and vertebrates
(Hoffman et al., 1999;
Medzhitov et al., 1997
;
Williams et al., 1997
;
Yang et al., 1998
). However,
only a subset of Drosophila TLRs induce defensive responses. TLR
family members appear divided into clans that function in innate immunity or
that fulfill developmental needs (Tauszig
et al., 2000
). The capacity to control glycosylation could unite
the TLR family in support of a common cause, to produce appropriate spatial
and temporal patterns of cell-specific glycosylation. Expressed by immune cell
types that participate in tissue surveillance, TLRs are positioned to locally
influence cellular glycosylation in response to pathogen, thereby coupling
innate detection of non-self patterns with expression of protective glycans on
host cells. In addition, further analysis of the distribution and function of
TLRs may indicate that the constitutive maintenance of diverse tissue glycan
profiles is generally an active process in which glycan expression is
continually renewed or responsively modified by TLR-mediated signaling. In
mature tissues and in the embryo, the expression of glycans must be
orchestrated to coincide with the appearance of relevant carbohydrate binding
proteins that mediate cell adhesion and recognition
(Varki, 1993
;
Sharrow and Tiemeyer, 2001
;
Feinberg et al., 2001
;
Song and Zipser, 1995
;
Vyas et al., 2002
). Therefore,
broader mechanisms that impart specificity to cell-cell interactions are
likely to be revealed with further characterization of the pathway by which
tollo/toll-8 controls oligosaccharide expression.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A.,
Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A.,
Galle, R. F. et al. (2000). The genome sequence of
Drosophila melanogaster. Science
287,2185
-2195.
Allendoerfer, K. L., Durairaj, A., Matthews, G. A. and Patterson, P. H. (1999). Morphological domains of Lewis-X/FORSE-1 immunolabeling in the embryonic neural tube are due to developmental regulation of cell surface carbohydrate expression. Dev. Biol. 211,208 -219.[CrossRef][Medline]
Altmann, F., Kornfeld, G., Dalik, T., Staudacher, E. and Glossl, J. (1993). Processing of asparagine-linked oligosaccharides in insect cells. N-acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells. Glycobiology 3, 619-625.[Abstract]
Altmann, F., Schwihla, H., Staudacher, E., Glossl, J. and Marz,
L. (1995). Insect cells contain an unusual, membrane-bound
beta-N-acetylglucosaminidase probably involved in the processing of protein
N-glycans. J. Biol. Chem.
270,17344
-17349.
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J.,
Zhang, Z., Miller, W. and Lipman, D. J. (1997). Gapped BLAST
and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 25,3389
-3402.
Amado, M., Almeida, R., Schwientek, T. and Clausen, H. (1999). Identification and characterization of large galactosyltranferase gene families: galactosyltransferases for all functions. Biochim. Biophys. Acta 1473,35 -53.[Medline]
Anderson, K. V., Jürgens, G. and Nüsslein-Volhard, C. (1985). Establishment of dorsal-ventral polarity in the Drosophila embryo: genetic studies on the role of the Toll gene product. Cell 42,779 -789.[Medline]
Belvin, M. P. and Anderson, K. V. (1996). A conserved signaling pathway: The Drosophila Toll-Dorsal Pathway. Ann. Rev. Cell Dev. Biol. 12,393 -416.[CrossRef][Medline]
Blochinger, K., Jan, L. Y. and Jan, Y. N. (1990). Patterns of expression of Cut, a protein required for external sensory organ development, in wild-type and cut mutant Drosophila embryos. Genes Dev. 4,1322 -1331.[Abstract]
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.
Brand, A. H., Manoukian, A. S. and Perrimon, N. (1994). Ectopic Expression in Drosophila. InDrosophila melanogaster: Practical Uses in Cell and Molecular Biology , Vol. 44 (ed. L. S. B. Goldstein and E. Fyrberg), pp. 635-654. San Diego, CA: Academic Press.
Campos-Ortega, J. A. and Hartenstein, V. (1985). In The Embryonic Development of Drosophila melanogaster, pp. 3-84. Berlin: Springer-Verlag.
Cantera, R., Kozlova, T., Barillas-Mury, C. and Kafatos, F. C. (1999). Muscle structure and innervation are affected by loss of Dorsal in the fruit fly, Drosophila melanogaster. Mol. Cell. Neurosci. 13,131 -141.[CrossRef][Medline]
Carlow, D. A., Corbel, S. Y., Williams, M. J. and Ziltener, H.
J. (2001). IL-2, -4, and -15 differentially regulate O-glycan
branching and P-selectin ligand formation in activated CD8 T cells.
J. Immunol. 167,6841
-6848.
Chen, L., Zhang, W., Fregien, N. and Pierce, M. (1998). The her-2/neu oncogene stimulates the transcription of N-acetylglucosaminyltransferase V and expression of its cell surface oligosaccharide products. Oncogene 17,2087 -2093.[CrossRef][Medline]
Chiang, C. and Beachy, P. A. (1994). Expression of a novel Toll-like gene spans the parasegment boundary and contributes to hedgehog function in the adult eye of Drosophila.Mech. Dev. 47,225 -239.[CrossRef][Medline]
Dennis, J. W., Granovsky, M. and Warren, C. E. (1999). Glycoprotein glycosylation and cancer progression. Biochim. Biophys. Acta 1473,21 -34.[Medline]
Desai, C. J., Popova, E. and Zinn, K. (1994). A Drosophila receptor tyrosine phosphatase expressed in the embryonic CNS and larval optic lobes is a member of the set of proteins bearing the `HRP' carbohydrate epitope. J. Neurosci. 14,7272 -7283.[Abstract]
Dodd, J. and Jessell, T. W. (1985). Lactoseries carbohydrates specify subsets of dorsal root ganglion neurons projecting to the superficial dorsal horn of rat spinal cord. J. Neurosci. 5,3278 -3294.[Abstract]
Eldon, E., Kooyer, S., D'Evelyn, D., Duman, M., Lawinger, P.,
Botas, J. and Bellen, H. (1994). The Drosophila
18-wheeler is required for morphogenesis and has striking similarities to
Toll. Development 120,885
-899.
Fabini, G., Freilinger, A., Altmann, F. and Wilson, I. B.
(2001). Identification of core alpha 1,3-fucosylated glycans and
cloning of the requisite fucosyltransferase cDNA from Drosophila
melanogaster. Potential basis of the neural anti-horseradish peroxidase
epitope. J. Biol. Chem.
276,28058
-28067.
Feinberg, H., Mitchell, D. A., Drickamer, K. and Weis, W. I.
(2001). Structural basis for selective recognition of
oligosaccharides by DC-SIGN and DC-SIGNR. Science
294,2163
-2166.
Feizi, T. (1985). Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens. Nature 314, 53-57.[Medline]
Frohman, M. (1993). Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE. Met. Enzymol. 218,340 -356.
Halfon, M. S. and Keshishian, H. (1998). The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo. Dev. Biol. 199,164 -174.[CrossRef][Medline]
Hammond, C., Braakman, I. and Helenius, A. (1994). Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc. Natl. Acad. Sci. 91,913 -917.[Abstract]
Hashimoto, C., Hudson, K. L. and Anderson, K. V. (1988). The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell 52,269 -279.[Medline]
Hoffman, J. A., Kafatos, F. C., Janeway, C. A. and Ezekowitz, R.
A. (1999). Phylogenetic perspectives in innate immunity.
Science 284,1313
-1318.
Ip, Y. T. and Levine, M. (1994). Molecular genetics of Drosophila immunity. Curr. Opin. Genet. Dev. 4,672 -677.[Medline]
Jan, L. Y. and Jan, Y. N. (1982). Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and grasshopper embryos. Proc. Natl. Acad. Sci. USA 79,2700 -2704.[Abstract]
Katz, F., Moats, W. and Jan, Y. N. (1988). A carbohydrate epitope expressed uniquely on the cell surface of Drosophila neurons is altered in the mutant nac (neurally altered carbohydrate). EMBO J. 7,3471 -3477.[Abstract]
Klämbt, C., Jacobs, J. R. and Goodman, C. S. (1991). The midline of the Drosophila central nervous system: a model for the genetic analysis of cell fate, cell migration, and growth cone guidance. Cell 64,801 -815.[Medline]
Kopczynski, C. C., Davis, G. W. and Goodman, C. S. (1996). A neural tetraspanin, encoded by late bloomer, that facilitates synapse formation. Science 271,1867 -1870.[Abstract]
Kurosaka, A., Yano, A., Itoh, N., Kuroda, Y., Nakagawa, T. and
Kawasaki, T. (1991). The structure of a neural specific
carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish
peroxidase antiserum. J. Biol. Chem.
266,4168
-4172.
Leviten, M. W. and Posakony, J. W. (1996). Gain-of-function alleles of Bearded interfere with alternative cell fate decisions in Drosophila adult sensory organ development. Dev. Biol. 176,264 -283.[CrossRef][Medline]
Lindsley, D. L. and Zimm, G. G. (1992). InThe Genome of Drosophila melanogaster , p.1075 . San Diego, CA: Academic Press.
Lowe, J. B. (2001). Glycosylation, immunity, and autoimmunity. Cell 104,809 -812.[Medline]
Matthews, R. T., Kelly, G. M., Zerillo, C. A., Tiemeyer, M. and
Hockfield, S. (2002). Aggrecan glycoforms contribute to the
molecular heterogeneity of perineuronal nets. J.
Neurosci. 22,7536
-7547.
Medzhitov, R., Preston-Hurlburt, P. and Janeway, C. A. (1997). A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388,394 -397.[CrossRef][Medline]
Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger, R. S. and Vogt, T. F. (2000). Fringe is a glycosyltransferase that modifies Notch. Nature 406,357 -358.[CrossRef][Medline]
Patel, N. H. (1994). Imaging neuronal subsets and other cell types in whole mount Drosophila embryos and larvae using antibody probes. In Drosophila melanogaster: Practical Uses in Cell and Molecular Biology, Vol. 44 (ed. L. S. B. Goldstein and E. Fyrberg), pp. 445-487. San Diego, CA: Academic Press.
Paulson, J. C. and Colley, K. J. (1989).
Glycosyltransferases: structure, localization and control of cell
type-specific glycosylation. J. Biol. Chem.
264,17615
-17618.
Phillis, R. W., Bramlage, A. T., Wotus, C., Whittaker, A.,
Gramates, L. S., Seppala, D., Farahanchi, F., Caruccio, P. and Murphey, R.
K. (1993). Isolation of mutations affecting neural circuitry
required for grooming behavior in Drosophila melanogaster.Genetics 133,581
-592.
Qian, R., Chen, C. and Colley, K. J. (2001).
Location and mechanism of alpha2,6-sialyltransferase dimer formation. Role of
cysteine residues in enzyme dimerization, localization, activity and
processing. J. Biol. Chem.
276,28641
-28649.
Rajput, B., Shaper, N. L. and Shaper, J. H.
(1996). Transcriptional regulation of murine beta
1,4-galactosyltransferase in somatic cells. Analysis of a gene that serves
both a housekeeping and a mammary gland-specific function. J. Biol.
Chem. 271,5131
-5142.
Rubin, G. M., Hong, L., Brokstein, P., Evans-Holm, M., Frise,
E., Stapleton, M. and Harvey, D. A. (2000). A Drosophila
complementary DNA resource. Science
287,2222
-2224.
Sambrook, J., Fritsch, E. and Maniatis, T. (1989). In Molecular Cloning: A Laboratory Manual. Plainview, NY: Cold Spring Harbor Laboratory Press.
Seppo, A. and Tiemeyer, M. (2000). Function and
structure of Drosophila glycans. Glycobiology
10,751
-760.
Sharrow, M. and Tiemeyer, M. (2001).
Gliolectin-mediated carbohydrate binding at the Drosophila midline
ensures the fidelity of axon pathfinding. Development
128,4585
-4595.
Snow, P. M., Patel, N. H., Harrelson, A. L. and Goodman, C. S. (1987). Neural-specific carbohydrate moiety shared by many surface glycoproteins in Drosophila and grasshopper embryos. J. Neurosci. 7,4137 -4144.[Abstract]
Song, J. and Zipser, B. (1995). Targeting of neuronal subsets mediated by their sequentially expressed carbohydrate markers. Neuron 14,537 -547.[Medline]
Stathopoulus, A. and Levine, M. (2002). Dorsal gradient networks in the Drosophila embryo. Dev. Biol. 246,57 -67.[CrossRef][Medline]
Sun, B. and Salvaterra, P. M. (1995) Characterization of Nervana, a Drosophila melanogaster neuron-specific glycoprotein antigen recognized by anti-horseradish peroxidase antibodies. J. Neurochem. 65,434 -443.[Medline]
Tauszig, S., Jouanguy, E., Hoffman, J. A. and Imler, J. L.
(2000). Toll-related receptors and the control of antimicrobial
peptide expression in Drosophila. Proc. Natl. Acad. Sci.
USA 97,10520
-10525.
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[Medline]
Tsuda, M., Kamimura, K., Nakato, H., Archer, M., Staatz, W., Fox, B., Humphrey, M., Olson, S., Futch, T., Kaluz, V., Siegfried, E., Stam, L. and Selleck, S. B. (1999). The cell-surface proteoglycan Dally regulates Wingless signaling in Drosophila.Nature 400,276 -280.[CrossRef][Medline]
Turkington, R. W., Brew, K., Vanaman, T. C. and Hill, R. L.
(1968). The hormonal control of lactose synthetase in the
developing mouse mammary gland. J. Biol. Chem.
243,3382
-3387.
Varki, A. (1993). Biological roles of oligosaccharides. Glycobiology 3, 97-130.[Abstract]
Vyas, A. A., Patel, H. V., Fromholt, S. E., Heffer-Lauc, M.,
Vyas, K. A., Dang, J., Schachner, M. and Schnaar, R. L.
(2002). Gangliosides are functional nerve cell ligands for
myelin-associated glycoprotein (MAG), an inhibitor of nerve regeneration.
Proc. Natl. Acad. Sci. USA
99,8412
-8417.
Wagers, A. J. and Kansas, G. S. (2000). Potent
induction of alpha(1,3)-fucosyltransferase VII in activated CD4+ T cells by
TGF-beta 1 through a p38 mitogen-activated protein kinase-dependent pathway.
J. Immunol. 165,5011
-5016.
Wang, X., Sun, B., Yasuyama, K. and Salvaterra, P. M. (1994). Biochemical analysis of proteins recognized by anti-HRP antibodies in Drosophila melanogaster: identification and characterization of neuron specific and male specific glycoproteins. Insect Biochem. Mol. Biol. 24,233 -242.[CrossRef][Medline]
Whitlock, K. E. (1993). Development of
Drosophila wing sensory neurons in mutants with missing or modified
cell surface molecules. Development
117,1251
-1260.
Williams, M. J., Rodriguez, A., Kimbrell, D. A. and Eldon, E.
D. (1997). The 18-wheeler mutation reveals complex
antibacterial gene regulation in Drosophila host defense.
EMBO J. 16,6120
-6130.
Yang, R. B., Mark, M. R., Gray, A., Huang, A., Xie, M. H., Zhang, M., Goddard, A., Wood, W. I., Gurney, A. L. and Godowski, P. J. (1998). Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling. Nature 395,284 -288.[CrossRef][Medline]
Yuki, S., Inouye, S., Ishimaru, S. and Saigo, K. (1986). Nucleotide sequence characterization of a Drosophila retrotransposon. Eur. J. Biochem. 158,403 -410.[Abstract]