Molecular and Cellular Biology, August 2001, p. 5688-5697, Vol. 21, No. 16
Howard Hughes Medical Institute and
Department of Genetics, Harvard Medical School, Boston,
Massachusetts 02115
Received 5 March 2001/Returned for modification 6 April
2001/Accepted 23 May 2001
The neurogenic gene brainiac was first isolated in
Drosophila melanogaster, where it interacts genetically
with members of the Notch signaling cascade. We have isolated a murine
homologue of the Drosophila brainiac gene and delineated
its highly specific expression pattern during development and adult
life. We find particularly strong expression in the developing central
nervous system, in the developing retina, and in the adult hippocampus. Targeted deletion of mouse Brainiac 1 expression leads to
embryonic lethality prior to implantation. Null embryos can be
recovered as blastocysts but do not appear to implant, indicating that
mouse Brainiac 1, likely a glycosyltransferase, is crucial for very early development of the mouse embryo.
The Notch signaling pathway has been
implicated in cell fate decisions in a variety of developmental
contexts in Drosophila melanogaster, Caenorhabditis elegans,
and vertebrates (4, 22, 52). Notch-dependent
signaling events include the induction of cell fates by cell-cell
interaction (a process termed "lateral specification"), as well as
the formation and maintenance of epithelial cell layers.
Notch encodes a transmembrane receptor that binds to the
membrane-bound ligands Delta and Serrate. Upon ligand
binding, Notch is cleaved by an unknown mechanism and an
intracellular portion of the molecule translocates together with the
Suppressor of Hairless [Su(H)] gene product to the nucleus, where the
protein complex acts as a transcriptional activator (34,
50). Genetic evidence from Drosophila indicates that
the genes of the Enhancer of split [E(spl)]
complex, which encode basic helix-loop-helix proteins, are targets of
the Notch signaling cascade (6, 13, 31, 37). Homologues of
Notch signaling molecules have been isolated in Xenopus
laevis, mice, and humans and demonstrate a high degree of
conservation of the Notch signaling cascade (23, 35, 45).
As in Drosophila, Notch signaling in vertebrates is involved
in cell fate specifications in a variety of developmental processes.
The neurogenic gene brainiac has been isolated in
Drosophila in screens for female sterile or larval lethal
mutations and has been shown to be involved in lateral specification
and epithelial morphogenesis (21). brainiac
mutant flies produce offspring with a neural hyperplasia and epidermal
hypoplasia reminiscent of hypomorphic Notch, Delta, or
E(spl) alleles, and genetic evidence indicates that
brainiac acts in the same genetic pathway as
Notch (21). However, in contrast to
Notch loss-of-function mutations, brainiac mutant
flies do not exhibit altered cell fate specifications during
oogenesis or development of the peripheral nervous system, suggesting that the brainiac gene product is only involved
in a subset of Notch signaling events in Drosophila.
Loss-of-function brainiac mutations in Drosophila
cause follicular epithelial cells surrounding the oocyte to lose their
epithelial morphology and to acquire a mesenchyme-like shape (18,
19), a phenotype also associated with hypomorphic
Notch alleles (53). The follicular epithelium
shows loss of epithelial polarity and aberrant cytoskeletal architecture in brainiac mutant flies, and cells accumulate
in multiple layers at the posterior end of the oocyte.
brainiac mutant flies frequently have discontinuities in the
follicular epithelium, presumably caused by a failure of follicle cells
to migrate over the top of or adhere to germ cells during early
follicular development. brainiac has been shown to act cell
nonautonomously in Drosophila, suggesting that brainiac
acts on proteins on their transport through the secretory pathway or
that brainiac itself is secreted.
In addition to its relationship with Notch signal transduction,
brainiac has also been shown to cooperate with the epidermal growth factor (EGF) receptor pathway during morphogenesis of the follicular epithelium in the Drosophila ovary (19,
21). brainiac mutant flies show aberrant
dorsal-ventral patterning during oogenesis and show genetic interaction
with gurken (the Drosophila homologue of
transforming growth factor Genetic evidence indicates that brainiac shares features
with fringe, a gene involved in defining dorsal-ventral
boundaries during formation of the Drosophila wing and the
vertebrate limb (20, 29, 36, 46). Both fringe and
brainiac are involved in regulating a subset of
Notch-dependent signaling events and also possess
significant sequence homology to glycosyltransferases, suggesting that
glycosylation of either Notch or its ligands modulates their
interactions (11, 25, 41, 56). Recent evidence shows convincingly that Drosophila and vertebrate Fringe proteins
are indeed glycosyltranferases modifying Notch itself and that
this glycosylation event modulates Notch-Delta interactions (9, 40).
Evidence from Drosophila shows that brainiac is
an important modulator of signaling and/or cell adhesion events during
embryonic development of the fly. In this report, we describe the
functional characterization of a putative murine homologue of the
Drosophila brainiac gene. Mouse Brainiac 1 is
expressed in the central nervous system (CNS) during development and
adult life. We eliminated Brainiac 1 expression by targeted
deletion in embryonic stem (ES) cells and show that Brainiac
1 is essential for preimplantation development of the mouse embryo.
Isolation of murine brainiac cDNA and genomic clones
and construction of a targeting vector.
To isolate a murine
homologue of the Drosophila brainiac gene, we screened the
EST database for vertebrate homologues using the BLAST algorithm. EST
clone yr97d07.r1 (Genome Systems), derived from a human spleen and
kidney library, possessed the highest sequence homology (at the protein
level) to Drosophila brainiac of any vertebrate sequence
found in the database at that time. The full-length cDNA insert of this
clone was used to screen a mouse 17.5-day-postcoitus (d.p.c.) embryonic
library (Clontech) using standard procedures.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.16.5688-5697.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
A Murine Homologue of the Drosophila
brainiac Gene Shows Homology to Glycosyltransferases and Is
Required for Preimplantation Development of the Mouse
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
[TGF-]), and the EGF
receptor (19, 21).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Targeted disruption of mouse Brainiac in ES cells and generation of Brainiac chimeric mice. A targeting vector (pmbrn11) was constructed in pOSdupdel (a gift kindly provided by O. Smithies), which contains an MCl-neo cassette (flanked by LoxP sites) for positive selection and an outside PGK-TK cassette for negative selection with FIAU. Adjacent genomic HincII fragments of 6 and 2 kb were ligated into the blunted XhoI site and the HpaI site, respectively, of the vector. This construct inserts the PGK-neo cassette into the Brainiac 1 coding sequence 140 bp 3' of the start codon, with transcription of neo and Brainiac 1 occurring in the same direction.
TC1 ES cells derived from 129/SvEv mice were electroporated with NotI-linearized pmbrn11 and selected with G418 and FIAU as described previously (14). G418- and FIAU-resistant clones were isolated and propagated in 24-well tissue culture dishes. Genomic DNA was isolated from these clones as described elsewhere (15) and sceened for targeting by digestion with EcoRI and AscI, followed by Southern blot analysis using standard conditions. Blots were hybridized with a 1-kbp BanI-AccI fragment (isolated from a BAC subclone) located 3' of the genomic region used to construct the targeting vector. After screening 230 G418- and FIAU-resistant colonies, we isolated one ES cell clone showing proper genomic rearrangement at the mouse Brainiac 1 locus. This positive ES cell clone was microinjected into C57BL/6J blastocysts, which were subsequently transferred into pseudopregnant Swiss Webster foster mothers (Taconic). We obtained two male and one female high-grade chimeric mice, as judged by agouti coat color. Chimeras were mated to 129/SvEv or NIH Bl/SW mice (Taconic), and germ line transmission was confirmed by Southern blot analysis. As expected, the two male chimeric mice showed germ line transmission of the introduced Brainiac 1 mutation, while the female chimera did not. Heterozygote offspring from this F1 cross were intercrossed to derive the mouse colony. PCR genotyping was performed using the following primers: mbrn1koF (5'-GGT GAT ATG GTA CCT CAG CCT CCC CCA CTA C-3'), mbrn1koR (5'-GTG AGG TCA CCA GGA TGA CCA GGA ATG GG-3'), and neoR (5'-AAT GAC AAG ACG CTG GGC GGG GTT TGC TCG-3'). Reactions were performed with Taq polymerase (Boehringer Mannheim) in 50-µl total volume, with all three primers simultaneously present at 200 nM each. Primary denaturation was performed at 94°C for 5 min, followed by 40 cycles of 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min. Reaction products were analyzed on 2.2% agarose gels. The wild-type allele (product mbrn1koF-mbrn1koR) was expected to give a PCR product of 166 bp, while the mutant allele (product mbrn1koF-neoR) was expected to give a PCR product of 280 bp.Isolation and genotyping of bastocysts. To isolate blastocysts, female Brainiac 1 heterozygote animals were superovulated with pregnant mare serum (PMS) and human chorionic gonatotrophin (HGG) and mated to Brainiac 1 heterozygote males. Blastocysts were isolated at 3.5 d.p.c. by flushing the uterus with CMRL-1066 medium (Gibco) containing 10% heat-inactivated fetal calf serum. Blastocysts were then cultured in 24-well tissue culture dishes at 37°C in CMRL-1066-10% heat-inactivated fetal calf serum-10x MEM Nonessential Amino Acids (Gibco), 10 mM L-glutamine (PenStrep; Gibco).
For genotyping, blastocysts were transferred to PCR tubes and lysed in 10 ml of 100 mM Tris (pH 8.0)-5 mM EDTA-0.2% sodium dodecyl sulfate (SDS)-200 mM NaCl-220 mg of proteinase K (Boehringer Mannheim) per ml overnight at 50°C. The proteinase K was heat-inactivated at 94°C for 159 min. Then, 2 µl of lysate was subjected to 50 cycles of PCR using the primers and conditions described above. PCR products were analyzed on 2.2% agarose gels. For blastocysts at stage 3.5 d.p.c., two separate reactions were performed using primer pairs mbrn1koF-mbrn1koR and mbrn1koF-neoR, respectively. PCR products were visualized by Southern blot hybridization using the following probes: the 836-bp mouse Brainiac 1 cDNA and a 280-bp mutant PCR product amplified from the targeting vector with mbrn1koR and neoR.Northern blot analysis. Total cytoplasmic RNA was isolated from cell lines or tissues using RNA STAT (Tel-Test, Inc.), and mRNA was prepared using magnetic poly(T) beads according to the manufacturer's protocols (Boehringer Mannheim). Next, 2 µg of mRNA was loaded on to a 1% agarose gel containing formaldehyde. After electrophoresis, the mRNA was blotted onto GeneScreen Plus membranes (NEN) and hybridized using standard procedures (5). A murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) full-length cDNA was used as a loading control.
In situ hybridization. For in situ hybridizations on tissue sections, tissues or embryos were fixed overnight in 4% formaldehyde. For P10 and adult brains, animals were perfused first with ice-cold phosphate-buffered saline (PBS) and then with 4% formaldehyde prior to fixation. After fixation, tissues were dehydrated in ethanol and mounted in paraffin. Samples were sectioned on a microtome (Reichert-Jung; 8-µm sections) and layered on glass microscope slides. Sections were dewaxed with xylene, treated for 7.5 min with proteinase K (20 mg/ml), and postfixed with 4% formaldehyde. Hybridizations were performed in 50% formamide-20 mM Tris (pH 7.4)-10% dextran sulfate-1× Denhardt's solution-10 mM dithiothreitol (DTT)-0.5 mg of yeast tRNA per ml. 35S-labeled riboprobes were added to the hybridization solution at 5 × 104 cpm/µl, and hybridizations were performed overnight at 52°C. After hybridization, slides were washed first with 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)- 10 mM DTT at 50°C and then with 2× SSC-50% formamide-10 mM DTT at 65°C and were finally treated with 20 µg of RNase A per ml for 30 min at 37°C. Slides were then dehydrated with ethanol and finally developed in NTP-2 photo emulsion (Kodak). Times of exposure were estimated from signal intensities on Cornex films after exposure overnight. Slides were developed using Kodak developer and counterstained with either hematoxylin and eosin (ovaries and embryos) or toluidine blue (brain sections) using standard procedures.
Control hybridizations were performed using a sense probe to Brainiac 1. In no case could a signal above background be detected (data not shown). For whole-mount in situ hybridizations, embryos were dissected from pregnant wild-type animals (FVB; Taconic) at various time points of pregnancy (8.5, 9.5, 10.5, and 12.5 d.p.c.) and fixed overnight in 4% paraformaldehyde at 4°C. After fixation, embryos were washed with PBS-0.1% Tween 20 (PBT) several times at 4°C and then dehydrated in a series of methanol-PBT. After incubation overnight in methanol, the embryos were rehydrated in a series of methanol-PBT, bleached with 6% hydrogen peroxide, treated with 10 µg of proteinase K (Boehringer Mannheim) per ml for 15 min at room temperature, and washed with 2 mg of glycine per ml in PBT for 10 min at room temperature. Embryos were then postfixed with 4% paraformaldehyde and 0.2% glutaraldehyde in PBT for 10 min, washed with PBT, and prehybridized in 50% formamide-5× SSC (pH 4.5)-1% SDS-50 µg of yeast RNA (Boehringer Mannheim) per ml-50 µg of heparin per ml for at least 1 h at 70°C. Mouse Brainiac 1 riboprobes were digoxigenin (DIG)-labeled using T7 and T3 RNA polymerases (DIG RNA Labeling Kit; Boehringer Mannheim) and purified using ethanol precipitation. Embryos were then hybridized in 50% formamide, 5× SSC (pH 4.5), 1% SDS, 50 µg of yeast RNA (Boehringer Mannheim) per ml, and 50 µg of heparin per ml overnight at 70°C. After hybridization, embryos were washed in 50% formamide-5× SSC (pH 4.5)-1% SDS and blocked in 10% sheep serum-TBST, and transcript was detected using an anti-DIG antibody (Boehringer Mannheim) and nitroblue tetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate) staining.| |
RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation of a mouse homologue of the Drosophila
brainiac gene.
We identified a putative murine homologue of
the Drosophila brainiac gene, which we termed mouse
Brainiac 1 (Mbrn 1), encoding a protein of 332 amino acids, with a predicted size of 39.4 kDa. The sequence isolated
here proved to be identical to the murine 
3-GalT-III gene
identified recently in an EST database search using human
-galactosyltransferase as the reference sequence (24).
Drosophila brainiac and the mouse homologue Brainiac
1 share several regions of high sequence similarity, although the overall sequence identity between the two proteins is only ca. 34%
(Fig. 1A). Interestingly, these domains
are also highly conserved among different murine
1,3-galactosyltransferase family members but are not found in
1,4- or 1,3-galactosyltransferases (Fig. 1B). Biochemical
analysis performed by others showed that mouse Brainiac 1 does indeed specifically catalyze 1,3 links between galactose and
N-acetylglucosamine (24). One of the conserved domains (domain 3, Fig. 1B) contains an (E/D)DVXXG motif that has also
been found in bacterial galactosyltransferases and may be associated
with a catalytic domain (56). These data suggest that
Drosophila brainiac and its homologues in higher organisms belong to the same family of 1,3-galactosyltransferases.
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Mouse Brainiac 1 is expressed in ovarian granulosa cells in a stage-specific manner. Using in situ hybridization on formalin-fixed sections of murine ovaries, we found mouse Brainiac 1 to be specifically expressed in follicular granulosa cells (Fig. 2B). Interestingly, expression of mouse Brainiac 1 appeared to be highly dependent on the stage of development of the follicle. While follicles containing only a single layer of tightly adhered granulosa cells did not express Brainiac 1, very high expression was exhibited in follicles at later developmental stages in which granulosa cells adhere less tightly to each other (Fig. 2B). Since the Drosophila brainiac gene has been shown to be important in establishing cell adhesion between follicular epithelial cells and germ cells during oocyte development, it is possible that mouse Brainiac 1 has a similar function during murine follicular development and modulates the adhesion properties of granulosa cells.
Expression of mouse Brainiac 1 during development. To establish the temporal and spatial distribution of mouse Brainiac 1 transcript during embryogenesis and adult life, we performed in situ hybridization on paraffin sections of mouse embryos and tissues.
In 11.5-d.p.c. embryos, we failed to detect a specific signal above background (data not shown). At day 12.5 of gestation, Brainiac 1 transcript could be detected in all four ventricles of the developing brain. At this stage of development, Brainiac 1 transcript was present in the ventricular zone, as well as in the mantle zone, although the signal appeared to be higher in the outer layers of the developing ventricles (Fig. 3A and B). An equivalent expression pattern in the developing CNS was detected at 14.5 d.p.c. (data not shown). This observation suggests that mouse Brainiac 1 is primarily associated with postmitotic cells during the development of the CNS. Thus, mouse Brainiac 1 expression appears to be upregulated when neuroepithelial cells exit the cell cycle and migrate toward the outer layers of the developing brain. This pattern is consistent with expression data from the P19 cell line, which has been used as an in vitro model for neurogenesis (33, 42). P19 cells express Brainiac 1 as undifferentiated precursor cells and at higher levels after retinoic acid-induced differentiation into neurons and glia (data not shown). In addition to this expression pattern in the CNS, we detected strong Brainiac 1 expression in the limb buds of developing embryos at 12.5 d.p.c. (Fig. 2C).
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Targeted disruption of mouse Brainiac 1 leads to
embryonic lethality prior to implantation.
Analysis of the murine
Brainiac 1 locus revealed that the entire coding region of
the gene is contained in a single exon of 996 bp. We disrupted the
murine Brainiac 1 gene by inserting a PGK-neo cassette 140 bp downstream of the start codon using homologous recombination in ES
cells (Fig. 4). Since no splicing is
anticipated in the stretch of RNA derived from this exon, this
insertion of the targeting cassette is expected to abolish expression
of the entire carboxy-terminal portion of the protein, leaving only 47 amino acids under the control of the endogenous promoter. This lesion
is expected to result in a null allele. ES cells transfected with the
targeting vector were selected with G418 for the presence of the neo
cassette and with FIAU for the loss of the thymidine kinase gene
present in the vector backbone. An ES clone with targeted disruption of
the mouse Brainiac 1 gene, as judged by Southern blot
analysis, was microinjected into blastocysts, and chimeric mice were
generated after transfer into pseudopregnant Swiss Webster foster
mothers. Offspring from the F1 generation obtained from matings of male chimeras to NIH BL/SW outbred mice were used to generate the mouse colony.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we report the functional characterization of a
putative murine homologue of the Drosophila brainiac gene.
The mouse gene described here encodes a protein with significant
overall amino acid sequence similarity to the Drosophila
brainiac protein. Interestingly, regions that show the highest
protein sequence conservation between the fly and mouse proteins are
also highly conserved in 1,3-galactosyltransferases but not in
1,4- or 1,3-galactosyltransferases, suggesting that brainiac in
Drosophila and Brainiac-like molecules in higher
organisms belong to a family of 1,3-galactosyltransferase enzymes
(12). This suggestion is further supported by the identity of the gene isolated in our screen (for vertebrate Brainiac
homologues) with a gene isolated in a database screen (for
galactosyltransferases) which encodes in vitro
1,3-galactosyltransferase activity (24). Biochemical
analysis revealed that mouse Brainiac 1 does indeed have
glycosyltransferase activity in vitro and catalyzes the formation of
galactose 1,3-N-actetylglucosamine structures
(24). No 1,4 linkage was detected in these in vitro
assays, suggesting that the enzymatic activity is highly specific for
1,3 links. A homologous gene has also been isolated in humans but,
surprisingly, does not show any glycosyltransferase activity in vitro
with the substrates tested (2). Since brainiac
acts cell nonautonomously in Drosophila, the putative
glycosyltransferase activity suggests that Brainiac might regulate
signaling events and/or cell adhesion processes through specific
glycosylation of cell-surface proteins.
Protein glycosylation has been shown to be an important modulator of
cell surface events in a variety of contexts, and mice carrying
targeted deletions in several genes for glycosyltransferases have been
described (16). While the loss of some
glycosyltransferases appears to lead to embryonic or perinatal
lethality, other enzymes of this family are not essential for
development and survival (10, 27, 39, 44). Malignant
transformation and metastatic potential have also been associated with
alterations in protein glycosylation. Ectopic expression of a
1,4-N-acetylglucosaminyltransferase in a B16 mouse
melanoma cell line suppresses the metastatic potential of these cells
in syngeneic mice (55); This malignant property has been
shown to depend on aberrant E-cadherin glycosylation (54),
indicating a link between glycosylation events and cell adhesion. In
addition, ectopic expression of a
1,4-N-acetylglucosaminyltransferase in PC12 cells has
been shown to disrupt NGF/Trk signaling by inhibiting receptor
dimerization but not receptor phosphorylation, causing inhibition of
neuronal differentiation in these cells. This observation indicates
that specific glycosylation events of receptors can modulate signaling
events (26). Interestingly, ectopic expression of mouse
Brainiac 1 does not interfere with NGF-induced
differentiation in PC12 cells, suggesting that glycosyltransferases
have very specific targets and modify specific biological events (B. Vollrath and K. Fitzgerald, unpublished observations).
As demonstrated in this report, mouse Brainiac 1 shows strong expression in the developing CNS and retina, two tissues where Notch signaling has been shown to regulate cell fate specifications during development. Although we find Brainiac 1 expression in undifferentiated neuroepithelial cells, Brainiac 1 expression appears to be upregulated in regions containing postmitotic, differentiated cells such as the ganglion cell layer in the retina or the outer layers of the developing ventricles. Notch receptors and its ligands are generally thought to be primarily expressed in undifferentiated neuroepithelial cells in the developing CNS and retina (7, 38). However, several genes involved in Notch signal transduction, such as Manic and Radical fringe are expressed in a pattern very similar to Brainiac 1 in the developing CNS (11). In addition, there is some evidence that Notch 1 is expressed in postmitotic neuronal cell populations in the ganglion cell layer of the developing retina and in the CNS and Notch 1 has been shown to regulate neurite outgrowth in differentiated, postmitotic neurons in vitro (1, 8, 47). In addition to the expression during retinal and CNS development, mouse Brainiac 1 shows strong expression in the limb buds during embryogenesis; in this tissue Notch receptors and their ligands are known to be expressed and function in pattern formation (48, 49).
Mouse Brainiac 1 also shows strong expression in the follicular granulosa cells of the ovary in a stage-dependent fashion. Granulosa cells that adhere tightly in an organized monolayer of epithelial cells show undetectable expression of mouse Brainiac 1, while cells that have lost their cell-cell adherence form less-organized multilayers in later-stage follicles and have a high level of Brainiac 1 expression. Since Drosophila brainiac has been implicated in regulating cell-cell adhesion during oogenesis in the fly, it can be hypothesized that mouse Brainiac 1-dependent glycosylation regulates the adhesive properties of follicular granulosa cells in the mouse.
Our results suggest that Brainiac-dependent glycosylation events
are essential for murine development. Loss of Brainiac-dependent glycosylation through targeted deletion of the gene leads either to
implantation failure or to embryonic death prior to implantation. This
observation suggests that glycosylation events by members of the
Brainiac protein family are highly specific, since other members of
this protein family are not able to compensate for the loss of
Brainiac 1 expression. The phenotype of Brainiac
1 null mice is also strikingly different from phenotypes of mice with targeted deletions of Notch or other components of the
Notch signaling cascade. These animals die at midgestation or later during development (30, 32, 51). However, mice with
targeted deletions of all murine Notch genes have not been
derived. The possibility that Brainiac-dependent glycosylation is
important for the function of all Notch receptors, thus
leading to the more severe phenotype in Brainiac 1 null mice,
cannot be ruled out. It is also possible that
Brainiac-dependent glycoslation is important for the
function of several different receptor types, thus leading to this very
severe phenotype of Brainiac 1 null mice. Since
brainiac shows genetic interaction with the EGF receptor and
the TGF- homologue gurken in Drosophila, this
pathway is an obvious candidate to be regulated by brainiac or
Brainiac-like molecules in Drosophila and higher organisms.
Genetic evidence in Drosophila indicates that
brainiac shares features with fringe, an
essential gene involved in pattern formation in the fly eye, wing, and
leg. Both genes encode molecules which appear to be involved in
regulating a subset of Notch signaling activities (20,
28). Fringe has been shown to modify the interaction of
Notch, with its ligands Delta and Serrate, by
potentiating the activating effects of Delta but repressing those
of Serrate and thereby giving specificity to the signal (17,
43). Sequence similarities between brainiac and fringe proteins,
which are very weak, can be detected with sensitive motif and profile
searches but not with standard alignment algorithms. Alignment of
Drosophila brainiac and fringe proteins suggests the
existence of a structural motif which is characteristic for procaryotic
and eucaryotic glycosyltransferases (41, 56). Since Notch
and its ligands Delta, Serrate, and Jagged are all glycoproteins,
specific glycosylation events mediated by Brainiac and Fringe
could conceivably modulate Notch-ligand interactions and thereby
regulate signaling events. Recent evidence in Drosophila and
mammalian systems shows that this is true for Fringe:
Fringe-dependent glycosylation of Notch modifies Notch signaling and modulates receptor-ligand interactions (9, 25, 40). It remains to be established whether Brainiac can act
in similar ways to modify signaling in this pathway. Although Fringe and Brainiac appear to catalyze similar 1,3 glycosylation events, the available in vitro data indicate that their substrate specificity is distinct suggesting distinct biological functions of these two
families of proteins (3, 24, 40).
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ACKNOWLEDGMENTS |
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We thank Oliver Smithies for the targeting vector pOSdupdel. In addition, we thank Ann Harrington for performing the blastocyst injections and for expert technical assistance with all mouse procedures, Monteserrat Michelmann for help with targeting ES cells and tissue culture procedures, Jan Pinkas and Nick Chester for helpful comments on the manuscript, and Katy Mclntyre for editing. We also thank all members of the Leder lab and the Harvard Medical School Department of Genetics for helpful comments and suggestions throughout the project.
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FOOTNOTES |
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* Corresponding author. Mailing address: Howard Hughes Medical Institute and Department of Genetics, Harward Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-7667. Fax: (617) 432-7944. E-mail: leder{at}rascal.med.harvard.edu.
Present address: Merck Research Laboratories, West Point, PA 19486.
Present address: Bristol Myers Squibb, Pennington, NJ 08530.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molecular and Cellular Biology, August 2001, p. 5688-5697, Vol. 21, No. 16
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.16.5688-5697.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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