1 Department of Biology, Denison University, Granville, OH 43023, USA
2 Department of Molecular Genetics and The Center for Molecular Neurobiology,
The Ohio State University, Columbus, OH 43210, USA
* Present address: College of Medicine, The Ohio State University, Columbus, OH
43210, USA
Present address: University Program in Genetics, Duke University, Durham, NC
27708, USA
Author for correspondence (e-mail:
liebl{at}denison.edu)
Accepted 14 April 2003
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SUMMARY |
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Key words: Neural development, Axon pathfinding, Abelson tyrosine kinase, Abl, amalgam, disabled, neurotactin, Drosophila melanogaster
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INTRODUCTION |
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One theme that has emerged from these genetic analyses is that the Abl
kinase may influence axon outgrowth by affecting cytoskeletal dynamics.
Dosage-sensitive genetic interactions affecting axon outgrowth have been found
between Abl and enabled (ena), with ena
being a dominant suppressor of the Abl mutant phenotype
(Gertler et al., 1990).
ena encodes an Abl substrate with multiple SH3-binding sites
(Gertler et al., 1995
) and was
the first identified member of the Ena/VASP family
(Lanier and Gertler, 2000
).
Its mammalian homolog Mena plays roles in axon guidance and cell migration by
affecting actin filament assembly (Bear et
al., 2000
; Bear et al.,
2002
; Gertler et al.,
1996
; Goh et al.,
2002
; Lanier et al.,
1999
; Renfranz and Beckerle,
2002
). Dosage-sensitive genetic interactions affecting axon
outgrowth have also been found between Abl and trio, with
trio being a dominant enhancer of the Abl mutant phenotype
(Liebl et al., 2000
).
trio encodes a protein with multiple domains, including an SH3 domain
and two guanine-nucleotide-exchange-factor domains
(Awasaki et al., 2000
;
Bateman et al., 2000
;
Liebl et al., 2000
;
Newsome et al., 2000
), which
can function to reorganize the actin cytoskeleton
(Newsome et al., 2000
).
Abl and the profilin homolog chickadee show dosage-sensitive
genetic interactions affecting axon outgrowth, with chickadee
dominantly enhancing a motoneuron phenotype in Abl mutants
(Cooley et al., 1992
;
Wills et al., 1999
). A protein
with similarities to neurofilaments, failed axon connections
(fax), has also been identified as a dominant enhancer of the
Abl mutant phenotype (Hill et
al., 1995
).
Abl has recently been shown to have multiple interactions with
robo signaling networks. robo encodes the transmembrane
receptor for Slit and is involved in regulating axon midline crossing
(Kidd et al., 1999;
Seeger et al., 1993
). Bashaw
et al. found that in a background sensitized by overexpression of Abl,
robo serves as a dominant enhancer with heterozygous mutations
strongly increasing inappropriate midline crossing in the CNS, leading to a
model whereby Robo is negatively regulated by Abl phosphorylation
(Bashaw et al., 2000
). Others
have also shown that Abl and Capt (a protein involved in actin cytoskeleton
dynamics) may be involved in restricting midline crossing in response to Slit
(Willis et al., 2002
).
Additionally, it has been shown that Abl may phosphorylate ß-catenin,
negatively regulating N-cadherin function in response to Slit-activated Robo
(Rhee et al., 2002
).
A dosage-sensitive genetic interaction that affects axon outgrowth between
Abl and disabled (dab) has been reported, with
dab dominantly enhancing the Abl mutant phenotype
(Gertler et al., 1989).
dab, which is the ortholog of murine Dab1
(Howell et al., 1997
) or
scrambler (Ware et al.,
1997
), encodes a tyrosine phosphorylated adaptor protein
containing a phosphotyrosine-binding (PTB) domain
(Gertler et al., 1993
;
Howell et al., 1997
). Work in
the murine system has shown Dab1 plays a role in neuronal migration
(Gallagher et al., 1998
;
Ware et al., 1997
).
This report now expands and modifies the cast of characters included in
axon extension networks involving Abl. We show that amalgam
(ama) and neurotactin (nrt) interact genetically
with Abl. ama encodes a secreted protein with three Ig-domains
(Fremion et al., 2000;
Seeger et al., 1988
), while
nrt encodes a transmembrane protein with a catalytically inactive
cholinesterase domain (Barthalay et al.,
1990
; de la Escalera et al.,
1990
; Hortsch et al.,
1990
) recently identified as the in vivo receptor for Amalgam
(Fremion et al., 2000
). We
have identified an unusual missense allele of ama generated in
genetic screens for strong dominant enhancers of the Abl mutant
phenotype. In addition, our molecular characterization of chromosomes carrying
dominant enhancers of the Abl mutant phenotype, originally identified
as dab alleles, has shown that these chromosomes in fact are mutant
for nrt. These mutations all show dosage-sensitive effects on axon
pathfinding in the Abl mutant background. As the binding of Amalgam
to Neurotactin promotes cell adhesion
(Fremion et al., 2000
), these
genetic interactions define an additional role for the Abl tyrosine kinase in
axon guidance.
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MATERIALS AND METHODS |
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Sequence analysis
In all cases genomic DNA was isolated from pupae or animals hemizygous for
the gene of interest. All amplifications for sequence analysis were carried
out using polymerase with proofreading activity (Advantage HF2, BD
BioSciences/Clontech, Palo Alto, CA). The ama ORF was amplified as
one 1.3 kb piece. The nrt ORF was amplified as four overlapping
fragments, each 800 bp. The dab ORF was amplified as eleven
fragments, each
850 bp. Not all of these fragments overlapped, as not all
of the two large introns (intron 1 and intron 4) of dab were
sequenced. Sequences of primers used are available on request. All fragments
showing a deviation from wild type were independently re-amplified and
re-sequenced.
RNAi and immunostaining
dsRNA was generated and injected into embryos as described
(Kennerdell and Carthew,
1998), with the exception that embryos were injected dorsally.
Primer sequences used to generate templates for in vitro transcription of
Abl, nrt and ama are available upon request. After
injection, embryos were raised at 18°C, and harvested at stage 14-15 for
mAb BP102 immunohistochemistry (Patel et
al., 1987
). mAbs BP102 and BP106 were obtained from the
Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA).
Anti-ß-galactosidase mAb (Promega, Madison WI) was used to detect
lacZ expression from enhancer-trap-containing balancer chromosomes to
distinguish the genotypes of the embryos.
S2 cell assays
All ama and nrt alleles were cloned into the pMET vector
(Bunch et al., 1988), under the
control of the metallothionine promoter. Stable S2 cell populations were
derived by co-transfecting with pPC4
(Elkins et al., 1990
) and
selecting for
-amanitin resistance. Fragments containing the mutations
associated with amaM109, nrtM100 and
nrtM221 were introduced into pMET-Ama, pMET-Ama-TM
(Fremion et al., 2000
) and
pMET-Nrt constructs using available restriction enzyme sites and confirmed by
DNA sequencing. For aggregation assays, conditioned media was generated by
inducing expression of pMET-Ama, pMET-AmaM109 or naïve S2
cells with 0.7 mM CuSO4 overnight. Ama-expressing cells were then
removed by centrifugation. The conditioned media was added to naïve S2
cells or cells transfected with different pMET-Nrt constructs. The addition of
conditioned media containing 0.7 mM CuSO4 initiated expression of
pMET-Nrt. Aggregation assays were conducted in six-well microtiter plates on a
rotary shaker at 80 rpm with
5x106 cells per ml.
Particles (cells and aggregates) were counted on a hemacytometer at
t=0, 4 and 8 hours. Cells transfected with pMET-Ama-TM and
pMET-AmaM109-TM were induced directly with 0.7 mM CuSO4
at the beginning of the aggregation assay. Accumulation of these
membrane-anchored forms of Ama was detected with mAb BP104, which recognizes
an epitope in the cytoplasmic domain of Nrg (Hortsch et al., 1995).
For cell pull-down assays, expression of Nrt was induced with overnight
exposure to 0.7 mM CuSO4. Nrt-expressing cells
(5x106 per ml) were recovered by low-speed centrifugation and
resuspended in Ama-conditioned media (see above). Cells were allowed to
aggregate for 60 minutes and then recovered by centrifugation. Cell pellets
were washed once with Schneiders media, re-pelleted and re-suspended in
Laemmli lysis buffer. Equivalent numbers of cells were used in all assays.
Equal volumes of cell lysates were loaded for immunoblot analysis. Nrt was
detected with mAb BP106 (Hortsch et al.,
1990). Ama was detected with rabbit anti-Ama antisera
(Seeger et al., 1988
).
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RESULTS |
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Null point-alleles of ama have never been described. In order to generate such alleles, we capitalized on our observation that a null allele of ama was not a dominant enhancer of the Abl mutant phenotype, and re-mutagenized the Abl1, amaM109 chromosome with EMS, screening for revertants of the dominant enhancement by amaM109 of the Abl mutant phenotype. Ten EMS-induced intragenic revertants of amaM109, all carrying the C46Y M109 mutation in addition to new alterations, have been recovered in this screen. Interestingly, nine of these revertants carry single missense mutations. We are currently characterizing the proteins encoded by these altered ama genes. However, one allele of ama, an R103C missense allele combined with a 29 bp deletion beginning in the P114 codon, was also generated. This is a null allele, as it can code for only the first 113 residues of Ama (with C46Y and R103C) followed by 54 novel residues due to the frameshift of the 29 bp deletion. We have named this revertant allele amaR1 (Fig. 2A).
We have found that 4% of the segments of embryos of the genotype Abl1, amaR1/Abl4 have defective commissures (n=176; Fig. 1D), and all of these embryos survive to pupation (Table 1). In both cases, these data were very similar to the characteristics of the Abl1, Df(3R)ama chromosome (2% defective segments, 92% survival to pupation) and very different from the parental Abl1, amaM109 chromosome (31% defective segments, 0% survival to pupation). We have observed better survival to adulthood of Abl1, amaR1/Abl4 animals as compared to Abl1, Df(3R)ama/Abl4 animals (Table 1), but as the Abl1, Df(3R)ama chromosome was generated by recombination and the Df(3R)ama deficiency is a multigenic deletion, this discrepancy may be attributable to differences in genetic backgrounds, and we have not investigated this observation further. The recovery of a null allele of ama in a screen for revertants of the dominant enhancement by amaM109 of the Abl mutant phenotype confirms our identification of the amaM109 allele as the causative mutation responsible for the dominant enhancement of the Abl mutant phenotype.
Biochemical and genetic characterization of the
amaM109 allele
Fremion et al. (Fremion et al.,
2000) have demonstrated that Ama binding to Nrt mediates cell:cell
adhesion. These authors have also shown that Ama proteins can self-associate,
suggesting a model whereby the binding of Ama to cell-surface Nrt, as well as
Ama:Ama homophilic binding somehow cooperate to promote cell:cell adhesion.
Similar to Fremion et al., we have employed two assays to examine the
biochemical behavior of Ama proteins. First, using S2 cells engineered to
express wild-type Nrt (see Materials and Methods), we have tested whether
media containing secreted Ama can mediate cell:cell adhesion. Cell adhesion
was quantified in these assays by counting total particles over time, with
particles defined as single cells and cell aggregates regardless of size. A
reduction in particle number indicated cell:cell adhesion, as free cells
aggregated into clusters. As part of this assay, we also indirectly monitored
Ama:Nrt binding by isolating cells exposed to Ama-containing media, and
testing for the specific binding of Ama to Nrt-expressing cells in a cell
pull-down assay. For our second assay, we generated Ama proteins fused to the
transmembrane and cytoplasmic domains of Neuroglian, thus creating a
membrane-anchored Ama (Fremion et al.,
2000
). By inducing the expression of these chimeric proteins, we
tested whether Ama:Ama interaction occurred by monitoring cell aggregation.
Both Ama+ and AmaM109 were tested in these assays.
While naïve S2 cells did not express Ama, after transfection with the appropriate expression plasmid both Ama+ and AmaM109 were produced as stable, secreted proteins at comparable levels (Fig. 2D, lanes 1, 2). Naïve S2 cells did not express Nrt (Fig. 4C, lane 1). Therefore, neither Nrt-expressing S2 cells exposed to naïve supernatant, nor naïve S2 cells exposed to Ama+ or AmaM109-containing supernatant showed aggregation (Fig. 2C), and naïve S2 cells did not bind Ama (Fig. 2D, lanes 3, 5). Ama+ did bind to Nrt-expressing S2 cells (Fig. 2D, lane 4), and supported their aggregation, with large aggregates of cells forming (Fig. 2B); total particle number was reduced by 48% over the 8 hour assay (Fig. 2C). AmaM109 also bound Nrt-expressing S2 cells (Fig. 2D, lane 6), but the aggregation it induced was clearly different from that seen with Ama+ with the aggregates that formed being small (Fig. 2B). In this case, total particle number was reduced by only 23% over the 8 hour assay (Fig. 2C). In addition, AmaM109-induced aggregates were less stable, disappearing after 24 hours, while Ama+-induced aggregates persisted beyond 24 hours (data not shown).
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Given the ability of AmaM109 to bind Nrt but mediate only limited cell:cell adhesion, and our observations that the amaM109 allele behaved as a dominant enhancer of the Abl mutant phenotype, while null alleles of ama did not, we wished to explore the genetic properties of the amaM109 mutation. The dominant activity of AmaM109 could be the result of dominant-negative effects. In this model, AmaM109 binding to Nrt exerts its influence by the exclusion of Ama+ binding, precluding normal Ama+-mediated cell:cell adhesion. Alternatively, the AmaM109 protein could have a more complex biochemical activity, actively producing phenotypic effects by occupying Nrt receptors. Using our amaR1 allele, we hypothesized that if amaM109 behaved strictly as a dominant-negative, the amaR1/Df(3R)ama and the amaM109/Df(3R)ama phenotypes would be similar. We found, however, a dramatic difference between the amaR1/Df(3R)ama and the amaM109/Df(3R)ama phenotypes in an Abl mutant background. Animals of the genotype Abl1, amaR1/Abl4, Df(3R)ama were found to have 23% of segments with defective commissures (n=255; Fig. 1E), while animals of the genotype Abl1, amaM109/Abl4, Df(3R)ama had 86% of segments with defective commissures (n=630; Fig. 1F). We therefore concluded that AmaM109 exerts phenotypic effects even in the absence of Ama+.
Elimination of Nrt strongly enhances the Abl mutant
phenotype
Ama is a secreted protein, while Abl is a cytoplasmic tyrosine kinase. The
transmembrane protein Nrt has been shown to be an in vivo receptor for Ama
(Fremion et al., 2000). Given
the strong genetic interaction between amaM109 and
Abl, and the unusual genetic character of
amaM109, we were curious to test for genetic interactions
between nrt and Abl. Although mutant alleles of nrt
exist (Speicher et al., 1998
),
nrt lies just proximal to Abl on chromosome three,
precluding the simple generation of an Abl-mutant,
nrt-mutant chromosome by recombination. We therefore elected to use
RNAi to generate Abl-null, Nrt-null embryos.
Embryos were injected with double-stranded RNA, fixed and mAb BP102 was
used to visualize axon tracts in the CNS. As shown in
Table 2 and
Fig. 3, elimination of Abl, Ama
or Nrt singly did not have a strong effect on the number of segments with
commissure defects. Although in buffer-injected control embryos, 3% of their
segments displayed commissure defects (n=833;
Table 2), Abl-null embryos
generated with RNAi showed commissure defects in 6% of their segments
(n=645; Table 2;
Fig. 3A). Ama-null embryos were
found to have 3% of their segments with commissure defects (n=159;
Table 2). In accordance with
previous data (Speicher et al.,
1998), Nrt-null embryos had mild CNS defects, with 8% of their
segments having commissure defects (n=383;
Table 2;
Fig. 3B). Eliminating Abl and
Ama together had a synergistic effect, with 32% of segments showing commissure
defects (n=350; Table
2). Eliminating Abl and Nrt together had an even more dramatic
effect, with 85% of segments showing commissure defects (n=443;
Table 2; Fig. 3C). There was also a
qualitative difference in the defects found, with the disruptions of the axon
scaffold resulting from the elimination of Abl and Nrt being much more severe
than with any other combination. Only in this background were breaks in
longitudinal axon tracts between anterior and posterior commissures observed.
In addition, many more disrupted segments were totally commissureless
(Fig. 3C).
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To explore the possibility further that some mutations attributed to dab could in fact be nrt alleles, we generated animals hemizygous for the dab and nrt alleles carried on these mutagenized chromosomes and sequenced both of these genes. For all five dab mutant chromosomes we recovered `escaper' animals over In(3L)std11 by supplying a single wild-type Abl transposon on the second chromosome. From the genomic DNA of these animals, we amplified and sequenced all exons and intron/exon boundaries of both dab and nrt.
For our sequence analysis of dab, we took the conceptual translation of the dab allele found under the Accession Number NM079395 (GenBank) as our wild-type benchmark. This would result in a Dab protein of 2224 residues. We found numerous polymorphisms in the dab alleles we sequenced compared with this wild-type benchmark. Some of these polymorphisms were common to all five alleles (N620T, V1061M, Y1241D, L1294Q, V1594A, D2089E). Some were specific to the individual parental chromosomes [Abl1: A1978V, A2175V; Df(3L)st-j7: S203A, Q543L, A557T, Q993K]. However, in no case did we find a unique mutation that would suggest a mutant dab allele had been generated on any of these chromosomes carrying dominant enhancers of the Abl mutant phenotype.
For our sequence analysis of nrt, we took the conceptual
translation of the nrt allele reported by Hortsch et al.
(Hortsch et al., 1990) as our
wild-type benchmark (GenBank Accession Number, X54999). The nrt
allele on both the parental Abl1 and the parental
Df(3L)st-j7 chromosome was found to be wild type.
From the three chromosomes that failed to produce mAb BP106 reactivity
[Df(3L)st-j7, dabM2;
Df(3L)st-j7, dabM29;
Abl1, dabM54] we found three independent null
alleles of nrt (Fig.
4A). The Df(3L)st-j7, dabM2
chromosome had a nonsense mutation, with the codon for L464 (TTG) being
changed to an amber stop (TAG). The Df(3L)st-j7,
dabM29 chromosome also had a nonsense mutation, with the codon
for Q267 (CAA) being changed to an ochre stop (TAA). The Abl1,
dabM54 chromosome was found to have an 11 bp deletion
beginning in the codon for I405. The resulting frameshift would lead to the
translation of two unique residues before a stop codon is reached. These
mutations are all consistent with the mutagens used to generate the dominant
enhancer mutations on these chromosomes, with the M2 and M29
alleles having been generated with EMS and the M54 allele having been
generated with X-rays (F. M. Hoffmann, personal communication).
As our molecular analysis of dab on the chromosomes that produced mAb BP106-reactivity (Abl1, dabM100; Abl1, dabM221) had failed to identify any telltale mutations, we also characterized the nrt alleles on these chromosomes. Interestingly, we found two independent missense mutations in nrt (Fig. 4A). The Abl1, dabM100 chromosome carried a V542D missense mutation, and the Abl1, dabM221 chromosome carried a G368E missense mutation. Both of these mutations map to the extracellular domain of Nrt, and both residues are conserved in the A. gambiae and D. pseudoobscura Nrt orthologs (data not shown). These mutations were consistent with the action of EMS that was used to generate the M100 and M221 mutations (F. M. Hoffmann, personal communication).
To explore these missense mutations further in nrt, we tested them in our S2 cell adhesion system. S2 cells expressing Nrt+, NrtM100 (V542D) or NrtM221 (G368E) were exposed to wild-type Ama. As shown in Fig. 4C (lanes 2-4), all three Nrt proteins were expressed at comparable levels. However, although Nrt+ expression mediated Ama+ binding and cell aggregation (Fig. 4B,C, lane 2), NrtM100 and NrtM221 behaved similar to naïve S2 cells exposed to Ama+-containing supernatant. Neither mutant Nrt protein mediated Ama+ binding (Fig. 4C, lanes 3, 4) and neither mutant Nrt protein mediated Ama-dependent cell:cell adhesion (Fig. 4B).
As different nrt null alleles had been created in different Abl mutant backgrounds, we were able to test the CNS phenotype of homozygous Abl mutant, nrt mutant embryos. As shown in Fig. 3F, embryos of the genotype Df(3L)st-j7, nrtM2/Abl1, nrtM54 showed disruption of the axon architecture, with 63% of segments showing clear commissure defects (n=187). This phenotype is consistent with, but somewhat less severe than the phenotype generated by RNAi elimination of Abl and Nrt (Fig. 3C).
Taken together, our molecular characterization of both dab and nrt from these chromosomes carrying dominant enhancers of the Abl mutant phenotype strongly suggested the causative mutations in all cases were in nrt and not dab. No mutations in dab were found, while three independent null alleles of nrt and two independent missense alleles that eliminate Nrt function were identified. In light of these molecular data, we propose that these dominant enhancers of the Abl mutant phenotype, previously identified as dabM2, dabM29, dabM54, dabM100 and dabM221 be renamed nrtM2, nrtM29, nrtM54, nrtM100 and nrtM221.
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DISCUSSION |
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In this study we report two novel dominant enhancers of the Abl
mutant phenotype: ama and nrt. The dosage-sensitive genetic
interactions between these genes and Abl provide unique information
regarding Abl signaling networks. We have identified five independent
nrt alleles that remove Nrt function. Three are null alleles
(nrtM2, nrtM29,
nrtM54), while two (nrtM100 and
nrtM221) are missense alleles that behave as protein nulls
(Fig. 4B,C). Thus, simply
reducing wild-type Nrt activity in an Abl-null background impairs
viability (Table 3), suggesting
Abl and Nrt lie within one or more common signaling networks. The fact that
these genetic combinations have clear effects on axon pathfinding
(Fig. 3D,E), strongly suggests
that at least one of these common signaling networks has its in vivo output in
the growth cone. This is confirmed by the severe axon guidance phenotype
produced by disruption of Abl and Nrt function through RNAi or homozygous
zygotic mutation (Fig. 3C,F).
Disruption of Abl and Nrt by zygotic mutation resulted in strong, but less
severe CNS phenotypes than RNAi, probably as a result of elimination of
maternally loaded Abl mRNA (Bennett and
Hoffmann, 1992).
It had been shown previously that Ama and Nrt functionally interact to
mediate cell:cell adhesion (Fremion et
al., 2000). Heterozygous null alleles of ama have no
detectable dominant effects on axon pathfinding in an Abl-mutant
background (Fig. 1C,D),
presumably because the biochemical activity of secreted Ama is not directly
associated with the cytoplasmic tyrosine kinase activity of Abl. However,
disruption of Abl and Ama by homozygous zygotic mutation
(Fig. 1F) or by RNAi techniques
(Table 2) did show clear
synergistic disruptions of the CNS architecture. As with Abl and Nrt, the
RNAi-induced phenotype was the more severe of the two, presumably because of
the elimination of maternally supplied Abl mRNA.
The identification of the unusual missense ama allele
amaM109 as a strong dominant enhancer of the Abl
mutant phenotype, affecting both viability and axon pathfinding
(Table 1,
Fig. 1B) strengthens our
conclusion that Ama, Nrt and Abl are functionally intertwined in the growth
cone. AmaM109, which alters a cysteine residue needed to stabilize
the first Ig domain of Ama (Fig.
2A), eliminates Ama homophilic adhesion but not the ability of
AmaM109 to bind Nrt (Fig.
2B-D), and this is probably responsible for its unique character.
The biochemical activity of this protein is clearly not wild type, as its
ability to support aggregation of Nrt-expressing S2 cells is impaired
(Fig. 2B,C). Interestingly,
Zhang and Filbin (Zhang and Filbin,
1998) have previously reported that destabilization of the
Ig-domain of myelin-specific protein Po results in a dominant-negative effect.
Po is a transmembrane protein with a single, extracellular Ig domain
stabilized by a disulfide bond between C21 and C98. Chinese hamster ovary
cells engineered to express Po showed strong homophilic adhesion. Coexpression
of a mutant Po, with an engineered C21A mutation, disrupted the aggregation
mediated by wild-type Po.
Genetically, the amaM109 allele phenocopies heterozygosity for nrt in the Abl1/Abl4 mutant background. Both genotypes result in 100% pre-pupal lethality (Table 1, Table 3), and both result in approximately one-third of embryo segments having defective commissures (Fig. 1B, Fig. 3D,E). Thus, it seems likely that, whatever its biochemical mode of action, the AmaM109 protein disables Nrt activity in a way that simply reducing the dose of wild-type Ama (by heterozygous null mutation) does not.
To understand the function of Nrt in the CNS better, Speicher et al.
(Speicher et al., 1998)
carried out an extensive genetic analysis, looking for cell adhesion molecules
(CAMs) that are functionally redundant to Nrt. This was achieved by generating
animals null for nrt and null for a variety of other CAM-encoding
genes in pair-wise combinations. Removal of Nrt does not result in a strong
CNS phenotype (Table 2,
Fig. 3B)
(Speicher et al., 1998
). Three
different genetic combinations showed synergistic interactions in the CNS;
nrt and neuroglian (nrg), nrt and
derailed (drl), and nrt and kekkon1
(kek1), with the nrt, nrg combination showing the most
profound synergy (Speicher et al.,
1998
). This work suggests the role of Nrt in CNS cell adhesion is
at least partially redundant to Nrg, Drl and Kek1. Interestingly, Elkins et
al. (Elkins et al., 1990
)
report that nrg and Abl have no genetic interaction when the
morphology of the CNS is assayed by mAb BP102 staining.
Whether Nrt-mediated adhesion provides novel inputs into Abl-mediated
signaling networks in the growth cone or whether Nrt-mediated adhesion
represents a novel output of the role of Abl in cytoskeleton dynamics can not
be determined by the genetic experiments we have carried out. Intriguingly,
Fremion et al. (Fremion et al.,
2000) report that deletion of the cytoplasmic region of Nrt
eliminates its ability to promote cell:cell adhesion. As many transmembrane
cell adhesion molecules require functional interactions with the actin-based
cytoskeleton (Petit and Thiery,
2000
), it is plausible that Ama:Nrt-mediated adhesion requires
interaction of the cytoplasmic region of Nrt with actin-based cytoskeleton
components. We are currently conducting molecular genetic screens to identify
protein:protein interactions involving the cytoplasmic domain of Nrt to
clarify this issue.
Our molecular and genetic characterization of nrt as a dominant
enhancer of the Abl mutant phenotype has shown that all five
mutations previously attributed to dab are nrt alleles
(Fig. 4A). How were these
mutations initially attributed to dab? Originally it was observed
that deletions that remove Abl as well as genes both proximal and
distal to Abl (Df(3L)st100.62,
Df(3L)st4, Df(3L)st-e5) or only genes
proximal to Abl (In(3L)std11), showed a
dominant enhancement of the Abl mutant phenotype
(Fig. 5)
(Henkemeyer et al., 1987). The
EMS-induced mutations M2 and M29 mapped tightly to
Df(3L)st-j7 and failed to complement
In(3L)std11, leading to the hypothesis that these
were alleles of a dominant enhancer gene lying proximal to Abl
(Gertler et al., 1989
). The
key observation that lead to dab seems to have been that
Df(3L)stE34 did not exhibit dominant enhancement of
the Abl mutant phenotype, while
Df(3L)st100.62 did show dominant enhancement of the
Abl mutant phenotype (Fig.
5) (Gertler et al.,
1989
). Molecular characterization of the proximal breakpoints of
these deletions showed Df(3L)st100.62 removed the
dab gene, while Df(3L)stE34 left
dab intact (Gertler et al.,
1993
), suggesting that dab was the gene proximal to
Abl responsible for the dominant enhancement of the Abl
mutant phenotype. However, in retrospect, the difference in genetic activity
between Df(3L)st100.62 and
Df(3L)stE34 can be accounted for by the difference
in the distal breakpoints of these chromosomes. Null mutations in fax
dominantly enhance the Abl mutant phenotype
(Hill et al., 1995
). On its
distal end Df(3L)st100.62 removes fax
(Hill et al., 1995
) while
Df(3L)stE34 leaves fax intact
(Fig. 5). Thus, although
nrtM2 and nrtM29 do lie proximal to
Abl, Df(3L)st100.62 has been shown to be wild type
for nrt (de la Escalera et al.,
1990
) and therefore the proximal breakpoint of
Df(3L)st100.62 does not uncover the dominant
enhancer gene identified by the M2 and M29 mutations.
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Our re-examination of the work of others in light of the fact that these
alleles are nrt mutations has revealed a startling fact: there may be
yet another dominant enhancer of the Abl mutant phenotype in the
73A-C region. As with Df(3L)st100.62, the dominant
enhancement of the Abl mutant phenotype reported for
Df(3L)st4
(Henkemeyer et al., 1987) is
likely to be due to its removal of the fax gene, distal to
Abl (Fig. 5). However,
Henkemeyer et al. (Henkemeyer et al.,
1987
) have also shown that Df(3L)st-e5
is a strong dominant enhancer of the Abl mutant phenotype. Based on
its reported cytology, Df(3L)st-e5 is wild-type for
fax (Fig. 5). The work
of de la Escalera et al. (de la Escalera
et al., 1990
) has shown the Df(3L)st-e5
chromosome is wild type for nrt. Thus, this deficiency apparently
uncovers another gene in the 73A-C region that can dominantly enhance the
Abl mutant phenotype. We are currently pursuing the identity of this
gene.
This report is not the first description of genetic interactions between
Abl and genes encoding cell adhesion molecules. Similar to the strong
phenotypic effects on CNS architecture that we have described in animals
homozygous mutant for Abl and nrt
(Fig. 3C,F), Elkins et al.
(Elkins et al., 1990) have
found that animals homozygous mutant for both Abl and
fasciclinI (fasI) show a mutant phenotype affecting midline
crossing of commissural axons. Unlike the present study, however, heterozygous
mutations in fasI had no effect on the Abl mutant phenotype.
The discovery of dosage-sensitive genetic interactions, such as that between
Abl and nrt, may indicate that the proteins encoded by these
two genes are biochemically linked. The continued search for dosage-sensitive
modifiers of the Abl mutant phenotype, and the continued biochemical
characterization of these modifiers will undoubtedly deepen our picture of the
molecular machinery guiding neuronal growth cones.
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ACKNOWLEDGMENTS |
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