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First published online March 6, 2009
doi: 10.1242/10.1242/dev.030254
1 Department of Biochemistry and Biophysics, The University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599, USA.
2 Department of Cell and Molecular Physiology, The University of North Carolina
at Chapel Hill, Chapel Hill, NC 27599, USA.
3 Curriculum in Neurobiology, The University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599, USA.
4 Department of Biology, The University of North Carolina at Chapel Hill, Chapel
Hill, NC 27599, USA.
* Authors for correspondence (e-mails: manzoor_bhat{at}med.unc.edu; steve_crews{at}unc.edu)
Accepted 4 February 2009
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MATERIALS AND METHODS
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Key words: Axon, Cell adhesion, Drosophila, Midline glia, Neurexin IV, Wrapper
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INTRODUCTION |
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) and extend processes that subdivide axon
bundles (Yoshioka and Tanaka,
1989). Like the floorplate cells, the midline glia (MG) of the
Drosophila CNS are centrally located and serve as a source of
secreted factors required for attracting axons to the midline and ensuring
that they do not recross (Garbe and
Bashaw, 2004). In each segment, the MG ensheath the anterior (AC)
and posterior (PC) commissures; each individual commissure is then further
subdivided into axon-containing regions separated by MG projections
(Noordermeer et al., 1998;
Stollewerk and Klambt, 1997).
The process of commissural axon ensheathment resembles vertebrate myelination,
requiring that the glia migrate towards, recognize, adhere to and completely
surround the axonal processes. These similarities make the Drosophila
MG an attractive system for studying the development and function of
neuron-glia interactions.
Initially, there are ten MG composed of two classes: anterior midline glia
(AMG) and posterior midline glia (PMG)
(Dong and Jacobs, 1997;
Kearney et al., 2004;
Wheeler et al., 2006). The AMG
initially consist of six cells. These cells migrate towards, make contact with
and ensheath the developing axon commissures through a series of stereotyped
movements and process extensions. On average, only three AMG closely contact
the commissural axons, resulting in their continued survival, while the
remaining AMG undergo apoptosis (Bergmann
et al., 2002). All PMG die via apoptosis, and do not contribute to
the mature MG (Dong and Jacobs,
1997; Sonnenfeld and Jacobs,
1995). Genetic studies have shown that the EGF, FGF and PVF
signaling pathways are involved in aspects of MG development
(Jacobs, 2000;
Learte et al., 2008). However,
members of these pathways are unlikely to mediate the adhesive interactions
that underlie MG-neuron interactions. Analysis of the immunoglobulin (Ig)
superfamily member wrapper showed that it is highly expressed in MG
and is required for MG-neuron adhesion, as well as for the proper ensheathment
and subdivision of the axon commissures
(Noordermeer et al., 1998).
One key issue is the identity of the binding partner on neuronal membranes
that functions with Wrapper in MG-neuron adhesion.
A strong candidate is Neurexin IV (Nrx-IV), which is
expressed throughout the CNS and encodes a transmembrane protein containing
four extracellular Laminin G domains and two EGF domains
(Baumgartner et al., 1996).
While distantly related to other vertebrate and invertebrate Neurexin
proteins, Drosophila Nrx-IV is orthologous to vertebrate Caspr
(paranodin; Cntnap1) (Banerjee et al.,
2006b). Caspr is localized to paranodal axo-glial junctions of
myelinated neurons, where it binds to the Ig superfamily proteins contactin
and neurofascin (Bhat, 2003;
Charles et al., 2002).
Drosophila Nrx-IV is also localized to septate junctions at axo-glial
interfaces, and interacts with Contactin and Neuroglian
(Banerjee et al., 2006a), which
is highly related to neurofascin.
Since Nrx-IV is expressed in neurons and binds to Ig superfamily
members, we tested the hypothesis that Nrx-IV and Wrapper physically interact
and mediate MG-neuron interactions. Using a sim-Gal4 UAS-tau-GFP
midline cell marker strain (Wheeler et
al., 2006) we were able to carefully examine midline cell
morphology, movement and axonal ensheathment during embryonic development.
Genetic analysis of Nrx-IV mutants revealed defects in MG-neuron and
MG-axon interactions that are identical to those observed with
wrapper mutants (Noordermeer et
al., 1998). Nrx-IV protein was highly localized to the interface
between MG and neuronal surfaces (both axon and cell body). The localization
of Nrx-IV on neuronal membranes was dependent on the presence of Wrapper, and
immunoprecipitation experiments demonstrated a physical interaction. Using
cultured Drosophila S2 cells, we showed that mixing wrapper-
and Nrx-IV-transfected cells resulted in cellular aggregation, and
this effect was dependent upon the presence of both proteins. As in embryos,
the Nrx-IV present in the aggregated cells was highly localized at sites of
cell-cell contact. Thus, Nrx-IV and Wrapper function as heterophilic adhesion
molecules that mediate MG migration and the ensheathment and subdivision of
commissural axons.
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),
arm-Gal4 (Sanson et al.,
1996), en-Gal4 (Ward
et al., 1998a), Nrx-IV4304
(Baumgartner et al., 1996),
Nrx-IV-GFP (CA06597) (Buszczak et
al., 2007; Laval et al.,
2008; Morin et al.,
2001), sim-Gal4 (Xiao
et al., 1996), slit-Gal4
(Scholz et al., 1997),
UAS-GFP-lacZ.nls (Shiga et al.,
1996), UAS-tau-GFP
(Brand, 1995),
UAS-wrapper (Noordermeer et al.,
1998) and wrapper175
(Noordermeer et al.,
1998).
In situ hybridization, immunostaining and immunoprecipitation
Embryo collection, in situ hybridization, immunostaining and
immunoprecipitation were performed as previously described
(Kearney et al., 2004;
Banerjee et al., 2006a).
Primary antibodies used were: mouse MAb BP102 (Developmental Studies Hybridoma
Bank, DSHB), rat anti-Elav MAb 7E8A10 (DSHB), mouse anti-En MAb 4D9
(Patel et al., 1989), chicken
anti-GFP (Upstate), rabbit anti-GFP Ab290 (Abcam), guinea pig anti-Lim3
(Broihier and Skeath, 2002),
rabbit anti-Nrx-IV (Baumgartner et al.,
1996), mouse anti-Nrt MAb BP106 (DSHB), guinea pig anti-Runt (East
Asian Distribution Center) (Kosman et al.,
1998), mouse anti-Wrapper MAb 10D3 (DSHB)
(Noordermeer et al., 1998) and
guinea pig anti-Wrapper. Generation of the guinea pig anti-Wrapper utilized a
6xHis-tagged fusion protein containing amino acids 245-444 of Wrapper as
immunogen. Midline cells were examined in abdominal segments A1-8. Owing to
the three-dimensional structure of the midline cells, it was difficult to
represent all relevant cells in a single focal plane; so, for clarity,
irrelevant portions of single images within a stack of confocal images were
subtracted before projections were generated.
Cell culture, RNAi and immunofluorescence
Cell culture and RNAi experiments were performed as described
(Rogers and Rogers, 2008).
wrapper (pAc-wrapper) and Nrx-IV
(pAc-Nrx-IV) open reading frames were PCR amplified from full-length
cDNA clones and cloned into the pAc-V5/His A vector (Invitrogen) providing
expression induced by the constitutively active Actin 5C promoter
(Han et al., 1989). For
immunofluorescence experiments, cells were plated onto poly-lysine-coated
coverslips 24 hours after transfection, fixed, and stained with antibodies
against Nrx-IV and Wrapper. All experiments were performed in duplicate and
more than 100 cells were analyzed for each sample. Control cells were
transfected with pMt-GFP to assess background aggregation. For cell
aggregation assays, S2 cells were transfected with either pAc-wrapper
or pAc-Nrx-IV by electroporation (Amaxa Nucleofector, Lonza). After
24 hours, the cells were resuspended, mixed, gently rocked on a Nutator for 1
hour, and plated onto poly-lysine-treated coverslips. For RNAi experiments,
cells were treated daily with 10 µg dsRNA for 6 days followed by
transfection with pAc-wrapper for 1 day. Negative controls were
conducted with dsRNA generated against pBluescript vector, whereas controls to
ensure that cells were competent for RNAi treatment were conducted with dsRNA
targeted to Rho1 (Rogers and
Rogers, 2008). Nrx1 and Nrx2 dsRNAs were generated by PCR of an
Nrx-IV cDNA with two primer sets: set 1,
5'-TAATACGACTCACTATAGGGGTCAATAATGGCATTAAATCGGTGCAAGCAGACG-3' and
5'-TAATACGACTCACTATAGGCCCAGTTCAGTGCTGTCTTTCATCACTCG-3'; set 2,
5'-TAATACGACTCACTATAGGGCACAGTACTATATTGCTGGTGGAAAGGACAAAAATGG-3'
and 5'-TAATACGACTCACTATAGGGCATCGGCAATGCGGAAGGTCACCACATTAC-3'.
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DISCUSSION
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; Wheeler et al.,
2008). In sim-Gal4 UAS-tau-GFP embryos, GFP is
localized to the cytoplasm of all midline cells - both neurons and glia
(Wheeler et al., 2006). When
examined in sagittal views, this allows visualization of the morphology of
each midline cell type during development. Our identification of specific
midline cell types employed immunostaining or in situ hybridization with more
than 90 validated cell type-specific reagents
(Wheeler et al., 2006;
Wheeler et al., 2008). In this
paper, we use this system to investigate the dynamics of MG development during
embryonic stages 12-17 in both wild-type and mutant embryos. MG were
identified by their distinct shape, relatively dorsal position within the CNS
and expression of wrapper, which is high in AMG, low in PMG, and
absent from neurons (Noordermeer et al.,
1998; Wheeler et al.,
2006). PMG were additionally identified by expression of
engrailed (en). Antibodies recognizing all neurons
(anti-Elav), their axons (MAb BP102), and the midline precursor 1 (MP1)
neurons (anti-Lim3) provided a comprehensive view of MG interactions with
nearby neurons and axons. At the beginning of CNS axonogenesis [stages 12/3 to
12/0; subdivisions of stage 12 according to Klambt et al.
(Klambt et al., 1991)],
commissural axons initially converged into a single axon bundle
(Fig. 1A,B). At stage 12/3, the
AMG resided in the anterior of the segment and had an elongated morphology as
they migrated toward the axon commissure
(Fig. 1A). Approximately three
AMG made contact with the anterior surface of the commissure while the
remaining AMG underwent apoptosis, in keeping with published observations
(Bergmann et al., 2002). At
stage 12/0, the AMG sent processes across both the dorsal and ventral surfaces
of the commissure (Fig. 1B). As
the commissure separated into AC and PC, the AMG membranes completely
ensheathed the AC (Fig. 1C). AC
ensheathment concluded with the movement of an AMG cell body between the
commissures (Fig. 1D). After
the AC was completely ensheathed, a single, dorsally located AMG migrated
across the dorsal surface of the PC during stages 15-16
(Fig. 1E). This AMG extended
processes posteriorly to ensheath the PC
(Fig. 1F). During stages 15-17,
the AMG also sent cytoplasmic projections into both the AC and PC that became
more elaborate as development proceeded
(Fig. 1E,F). Using electron
microscopy, Stollewerk and Klambt
(Stollewerk and Klambt, 1997)
showed that these cytoplasmic projections divide each commissure into distinct
subdomains.
Both the MP1 neurons and PMG were also in proximity to AMG and the
commissures. Their positions were constant relative to the migrating AMG. The
MP1 neurons remained in close contact with the ventral-most AMG from stages
11-17 (Fig. 1B-F; see Fig.
S1A-D in the supplementary material), and prior to commissure separation the
MP1 neurons were closely associated with the commissure along its ventral side
(Fig. 1B; see Fig. S1A in the
supplementary material). The PMG migrated dorsally and in an anterior
direction during stage 12, and at least one PMG abutted the posterior side of
the PC from stages 12-16 (Fig.
1A-E; see Fig. S1E-H in the supplementary material) before
undergoing apoptosis (Bergmann et al.,
2002; Sonnenfeld and Jacobs,
1995). The cell bodies of non-midline-derived neurons also made
extensive contacts with the AMG (see Fig.
4D) and, together with the PMG and MP1 neurons, they might play
important roles in AMG development. Overall, the view of MG development
described above is in general agreement with that described by others
(Jacobs, 2000), validating the
use of sim-Gal4 UAS-tau-GFP to study MG development. However, there
are some important differences in nomenclature and PMG migration (see
Discussion).
Nrx-IV mutants have disrupted MG-neuron interactions
During an earlier analysis of the role of Nrx-IV in septate
junctions (Banerjee and Bhat,
2007; Baumgartner et al.,
1996), a failure of commissure separation was observed in
Nrx-IV mutants (Fig.
2A,B). Previously, disruption of the commissures was correlated
with MG defects (Klambt et al.,
1991). To investigate a potential Nrx-IV midline
phenotype, Nrx-IV4304, a null allele, was used in
combination with sim-Gal4 UAS-tau-GFP to examine the dynamics of AMG
migration, axonal ensheathment and commissure subdivision. During stage 12,
the migration of the AMG and their juxtaposition to the unseparated commissure
were normal (Fig. 2C). Once the
AMG contacted the commissure, they sent processes across the AC as in the wild
type (Fig. 2D). However,
instead of ensheathing the AC, both the dorsally and ventrally located AMG
migrated past the AC towards the PC (compare
Fig. 2E,F with
Fig. 1C,D). Strikingly, the AMG
were frequently dissociated from the MP1 neurons residing at a more distant
and dorsal location away from the midline neurons
(Fig. 2H,I). This phenotype was
the most common defect observed in stage 17 Nrx-IV mutant segments
(Table 1). In stage 17
wild-type embryos, 3.1±0.4 AMG (n=33 segments) were present,
whereas in embryos with dissociated AMG this was reduced to 2.1±0.5 AMG
(n=28 segments). The reduction was likely to be due to the lack of
AMG ensheathment of the PC, and the corresponding inability of the AMG to
receive a sufficient axon-derived survival signal
(Bergmann et al., 2002).
Additional Nrx-IV mutant phenotypes were: (1) dissociation with
incomplete migration and ensheathment of the AC and PC
(Fig. 2J;
Table 1), and (2) a complete
absence of AMG (Fig. 2K;
Table 1). In all mutant
segments, the AMG failed to extend glial projections to subdivide either the
AC or PC (Fig. 2A,B; and
compare Fig. 2I with
Fig. 1F). The inability of the
AMG to properly migrate along the AC coupled with their inability to send
projections into the commissures indicated that Nrx-IV was required
for interactions between AMG and commissural axons. Furthermore, the
dissociation of AMG from the MP1 neurons indicated that interactions between
these two cell types are important for AMG positioning.
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). We argue that the Nrx-IV midline staining is due to
Nrx-IV neuronal expression and protein localization at MG-neuron
interfaces, and is absent from MG (or present at insignificant levels). There were two major locations at which Nrx-IV was concentrated (Fig. 3C): (1) along the axon commissures, and (2) at the boundaries between MG and neurons. At stages 12/0 to 13, Nrx-IV was found at the interface where the migrating AMG contacted the single, unseparated commissure (Fig. 3D). After commissure separation and ensheathment, Nrx-IV localized to the boundaries where commissures and AMG were juxtaposed and along the AMG projections that subdivided the commissures (Fig. 3E).
Nrx-IV was localized at detectable, but low, levels around the membranes of
neurons (Fig. 3B), and at high
levels at sites of contact between lateral CNS neurons and AMG
(Fig. 3F). Neurons also showed
Nrx-IV localization where they contacted PMG, but the accumulation was weaker
than with AMG (Fig. 3C). Nrx-IV
was highly localized to the contacts between AMG and the MP1 neurons
(Fig. 3G); this localization
was prominent from stage 12/3 until the MP1 neurons underwent apoptosis during
stage 17 (Miguel-Aliaga and Thor,
2004). To further investigate the subcellular localization of
Nrx-IV, we examined a protein-trap GFP fusion of Nrx-IV (Nrx-IV-GFP). The
localization of Nrx-IV-GFP strongly resembled endogenous Nrx-IV protein with
respect to cell type localization: it was present in neurons but absent or at
low levels in MG (Fig. 3H,I).
Although Nrx-IV-GFP localized at MG-neuron interfaces, it was less pronounced
than endogenous Nrx-IV. Conversely, the Nrx-IV-GFP was more cytoplasmic than
Nrx-IV and its localization was enhanced in the axon scaffold
(Fig. 3I). Neither Nrx-IV
immunostaining nor Nrx-IV-GFP was observed at appreciable levels at the
membranes of AMG or PMG that were not contacting neurons
(Fig. 3C,H). These data suggest
that Nrx-IV is expressed in neurons and not MG.
To test the requirement for Nrx-IV in neurons or glia, we used a
neural driver (elav-Gal4) and an MG driver (slit-Gal4) to
express UAS-Nrx-IV in a protein-negative
Nrx4304-null mutant and assayed Nrx-IV protein
localization and rescue of the Nrx-IV MG migration defects. In
elav-Gal4 rescue embryos, Nrx-IV was localized at the boundaries
between neurons and both AMG and PMG (compare Fig.
3J with
3B), similar to wild type.
Unlike in Nrx-IV mutants, in these rescue embryos AMG ensheathed the
AC by stage 15 and migrated between the AC and PC
(Fig. 3K-M;
Table 2). However, by late
stage 17, the AMG failed to ensheath the PC or to send projections into either
commissure (data not shown). Thus, misexpression of Nrx-IV using
elav-Gal4 was able to rescue the initial steps of MG migration, but
not the later aspects. We noted that Nrx-IV protein levels were lower in the
rescue embryos from stages 15 to 17 as compared with wild-type embryos (data
not shown), a result consistent with the decrease in elav-Gal4
expression reported previously (Lin and
Goodman, 1994). This might explain the lack of full rescue late in
development. slit-Gal4 rescue embryos did not show localization of
Nrx-IV at MG-neuron boundaries (see Fig. S1L,N in the supplementary material)
and the Nrx-IV mutant MG migration defects were not rescued either at
stage 15 or 17 (Table 2; see
Fig. S1L-O in the supplementary material). In summary, only when expressed in
neurons did Nrx-IV accumulate at MG-neuron interfaces and partially rescue the
Nrx-IV mutant phenotype.
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;
Wheeler et al., 2006). Unlike
Nrx-IV, wrapper in situ hybridization showed strong MG expression
(Fig. 4A). Wrapper protein was
first observed at stage 12/3 and was present throughout AMG migration and
ensheathment. The protein was uniformly localized on MG membranes
(Fig. 4B), including where they
contacted MP1 neurons (Fig.
4C), commissural axons (Fig.
4C,E), lateral CNS neuronal cell bodies
(Fig. 4D) and other MG
(Fig. 4B). At later stages,
Wrapper was present on AMG projections during commissure subdivision
(Fig. 4E).
Genetic analysis of wrapper175-null mutant embryos
revealed MG phenotypes that were identical to Nrx-IV mutant embryos,
and to the wrapper phenotypes reported previously using electron
microscopy (Noordermeer et al.,
1998). At stage 12, the AMG and PMG of wrapper mutants
migrated dorsally and contacted the commissures normally. Defects appeared
after the AMG contacted the AC. By stage 15, AMG failed to ensheath the AC,
and an AMG cell body was not interposed between the AC and PC
(Fig. 4F). The wrapper
mutant phenotype was highly penetrant: 99% of stage 17 segments possessed
defects (n=139) (Table
1). In 76% of mutant segments, AMG surrounded the AC but failed to
surround the PC (Fig. 4G), and
in the other 24% of mutant segments, the AMG failed to surround either the AC
or PC (Fig. 4H). In all
segments examined, the AMG were dissociated from the MP1s
(Fig. 4I) and failed to extend
projections to subdivide the commissures
(Fig. 4G). As in
Nrx-IV mutants, the number of AMG present during stage 17 was
reduced, from 3.1±0.4 (n=33) in wild type to 2.3±0.7
(n=14). Thus, wrapper is required for AMG migration,
commissural ensheathment and commissure subdivision. Phenotypes of Nrx-IV
wrapper double mutants were similar to, but more severe than, either
single mutant: the same percentage of segments showed defects as in the single
mutants, but more segments showed incomplete ensheathment of either the AC or
PC (Table 1). One
interpretation of the more severe double-mutant phenotype is that both Nrx-IV
and Wrapper might be interacting with additional proteins on the apposing
cell.
Nrx-IV membrane localization is dependent on Wrapper
In wild-type embryos, Nrx-IV protein was present at low levels throughout
the membranes of neuronal cell bodies and axons, but was highly concentrated
where they contacted AMG (Fig.
3B; Fig. 5A). The
similarity of the Nrx-IV and wrapper mutant phenotypes
coupled with the concentration of Nrx-IV opposite Wrapper+
MG membranes led us to hypothesize that the high-level membrane localization
of Nrx-IV was dependent on the presence of Wrapper on the apposing membranes.
To investigate this possibility, Nrx-IV localization was examined in
wrapper mutant and misexpression embryos. In wrapper
mutants, Nrx-IV was not concentrated at the contact points of neurons with MG,
but was instead distributed uniformly around neuronal cell bodies
(Fig. 5A,B) and axons
(Fig. 5C,D) at higher levels
than observed in wild-type embryos.
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Nrx-IV and Wrapper interact to induce cell adhesion in S2 cells
To test the hypothesis that Nrx-IV and Wrapper act as heterophilic cell
adhesion partners, S2 cells were used to assay cell adhesion
(Hortsch and Bieber, 1991).
Immunoblot analysis of S2 cell extracts revealed that Nrx-IV was abundantly
present as a 155 kDa protein (Fig.
6B); by contrast, Wrapper was undetectable (data not shown). S2
cells were transfected at high concentration with pAc-wrapper and
pAc-Nrx-IV. Transfection of Nrx-IV alone did not result in
the formation of aggregates (Fig.
6D), whereas transfection of wrapper alone resulted in
the formation of small aggregates (2-15 cells/aggregate)
(Fig. 6E). Mixing together of
Nrx-IV- and wrapper-transfected cells resulted in the
appearance of large aggregates (>100 cells/aggregate)
(Fig. 6F), indicating that
Nrx-IV and Wrapper bind in trans and mediate cell adhesion.
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To analyze Nrx-IV and Wrapper protein localization in the aggregates, we examined the small aggregates generated by transfection of wrapper alone. wrapper-transfected cells were readily identifiable by their prominent cortical labeling with anti-Wrapper antibody (Fig. 6G-I). The sites of cell-cell contact with Wrapper- cells displayed a pronounced recruitment of Nrx-IV into cortical patches (Fig. 6G-I); these were never observed in untransfected cells. We did not observe enrichment of Wrapper to Nrx-IV+ patches, and did not see Nrx-IV+ patch formation at sites where two Wrapper+ cells were in contact (Fig. 6H,I). This phenomenon was also observed in en-Gal4 UAS-wrapper embryos, in which Nrx-IV was absent from sites of contact between Wrapper+ cells (Fig. 5F). This result might be due to the ability of high levels of Wrapper to saturably bind pools of Nrx-IV intracellularly - this competition would not leave sufficient Nrx-IV to bind to Wrapper in adjacent cells and form observable Nrx-IV+ membrane patches. Alternatively, Nrx-IV-Wrapper intracellular interactions could inhibit Nrx-IV transport and assembly into the membrane, or promote its internalization or degradation. Overall, these data show that expression of wrapper causes membrane accumulation of Nrx-IV similar to that observed in the embryonic CNS.
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SUMMARY
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MATERIALS AND METHODS
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DISCUSSION
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), and show that both genes have identical MG phenotypes, a
likely outcome for two genes involved in heterophilic cell adhesion. The
Nrx-IV-Wrapper interactions occur between MG and three neuronal cell types or
structures: (1) MP1s, (2) cell bodies of lateral CNS neurons, and (3)
commissural axons. Together, these interactions control the position of MG and
the ensheathment and subdivision of commissures.
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MG-lateral CNS neuronal cell body interaction
Nrx-IV protein is present in most, if not all, CNS neurons. However,
protein levels are generally low. The exceptions are the neurons that flank
the MG. These cells show a strong accumulation of Nrx-IV at the interfaces
with MG. This indicates an aspect of midline cell biology not commonly
considered - that MG interact closely with adjacent lateral CNS neurons. This
might act to physically constrain migrating MG at the midline and restrict
their lateral movement. In this sense, the lateral CNS neurons, the MP1
neurons and axon commissures work together to construct the MG
cytoarchitectural scaffold. Alternatively, the adhesion between lateral CNS
neurons and MG might allow developmental signals to pass between these cell
types.
MG-commissural axon interaction
A key functional role of MG is their interaction with commissural axons,
and these interactions require complex MG movements and morphological changes.
The AMG extend cytoplasmic processes between the commissures, followed by an
AMG cell body. These MG structures effectively partition the AC from the PC.
Previously, it was proposed that commissure separation is caused by the
interposition of MG into the unseparated commissure
(Klambt et al., 1991).
However, we noted that in wrapper mutants, the AC and PC were well
separated, even though MG processes were commonly absent between the
commissures (Fig. 4F-H). It is
possible that in wrapper mutants, MG initially caused commissure
separation and then quickly retracted or underwent apoptosis, indicating that
MG function was required only transiently. Alternatively, commissure
separation could be independent of MG interposition and the MG partition
already-separated commissures. In contrast to wrapper mutants,
Nrx-IV mutants have poorly separated commissures. This difference is
most likely to reflect an additional function of Nrx-IV because: (1)
the MG phenotypes were similar between Nrx-IV and wrapper
mutants, (2) the mutants of each gene were null, (3) neither had a
recognizable maternal effect, and (4) Nrx-IV was more widely
expressed.
Throughout commissure ensheathment, axons have strong accumulations of Nrx-IV along their interface with the AMG. This suggests a continual requirement of Nrx-IV and Wrapper to mediate MG-axonal adhesion and is consistent with the wide variety of MG-axon adhesion defects observed in both Nrx-IV and wrapper mutants and the inability of elav-Gal4 UAS-Nrx-IV to rescue late Nrx-IV mutant phenotypes. By contrast, MG remained relatively well associated with each other, suggesting that neither wrapper nor Nrx-IV plays an important role in MG-MG adhesion.
MG projections also subdivide each commissure into discrete compartments.
Previous work employing electron microscopy proposed that the MG subdivided
each commissure into three dorsoventral regions
(Stollewerk and Klambt, 1997).
This subdivision also requires Nrx-IV and wrapper function
because Nrx-IV and Wrapper accumulated in the AMG commissural projections, and
the projections were absent in both Nrx-IV and wrapper
mutants (Noordermeer et al.,
1998). Both the organizing principles and the significance of
these commissural subdomains are unknown, and it remains to be determined
whether the MG are a cause of the subdivision or are filling in axonal regions
that are already subdivided.
Perspectives on MG migration
The view of MG migration presented here builds on previous work, but also
differs in several aspects. These include nomenclature, MG-neuron interactions
and PMG migration. Klambt et al. (Klambt
et al., 1991) proposed a model in which three pairs of MG (MGA,
MGM and MGP) arise in the anterior of the segment and, during migration,
separate and ensheath the AC and PC. The MGA and MGM migrate posteriorly and
ensheath the AC; the MGA ultimately resides anterior to the AC and the MGM
between the AC and PC. By contrast, the MGP migrate anteriorly from the
adjacent posterior segment and partially ensheath the PC. More recent
observations, including some from this paper, point toward a different view.
Analysis of 52 genes expressed in MG
(Kearney et al., 2004)
indicates that (to date) only two distinct MG cell types can be identified,
which we have termed AMG and PMG. There are six AMG in the anterior of the
segment (this class includes MGA and MGM, which, to our knowledge, cannot be
distinguished molecularly) and four PMG that reside in the posterior of the
segment and are identical to MGP in terms of gene expression. Of the six
initial AMG, only three survive (Bergmann
et al., 2002). These cells migrate posteriorly, ensheath both the
AC and PC, and elaborate projections into the commissures. By contrast, all
PMG die by stage 17 (Dong and Jacobs,
1997; Sonnenfeld and Jacobs,
1995), and therefore do not ensheath the PC. Initially, it was
proposed that PMG/MGP migrate from the adjacent posterior segment. In our
experiments, we see no evidence for this. Instead, PMG arise in the
En+ posterior of the segment and migrate anterodorsally toward the
commissure. Before undergoing apoptosis, a single PMG abuts the PC from the
posterior side. Thus, the PMG are positioned to influence commissure
development.
Neurexin IV and immunoglobulin superfamily protein interactions
The experiments described in this paper strongly support the view that
Nrx-IV and Wrapper directly bind and mediate cell adhesion. By contrast,
neither protein mediates homophilic cell adhesion
(Baumgartner et al., 1996;
Noordermeer et al., 1998).
Wrapper is an Ig superfamily protein, and experiments in both flies and
vertebrates indicate that Nrx-IV can bind to additional Ig superfamily
proteins. In Drosophila septate junctions, Nrx-IV forms a complex
with Contactin and Neuroglian
(Faivre-Sarrailh et al.,
2004), which are two Ig superfamily proteins. It was proposed that
Nrx-IV binds to Contactin at the membrane in a cis configuration
(Faivre-Sarrailh et al.,
2004), and that Contactin is required for proper Nrx-IV membrane
localization (Laval et al.,
2008). Contactin is present in the CNS, and might play a similar
role in neurons. It is unknown whether Nrx-IV binds Neuroglian or Contactin in
trans, similar to the mechanism proposed here for Nrx-IV-Wrapper binding.
However, at paranodal axo-glial junctions in mice, the Nrx-IV homolog Caspr
binds in cis to contactin, and in trans to the Neuroglian homolog neurofascin.
In summary, Nrx-IV binding to Wrapper indicates a general feature of Nrx-IV,
which is its ability to bind diverse Ig superfamily proteins.
One of the remarkable aspects of Nrx-IV is its strong membrane accumulation
at sites where neurons are apposed to MG. In one sense, this resembles the
accumulation of Nrx-IV in septate junctions. However, from a mechanistic
perspective the situation appears different. In septate junctions, Nrx-IV
membrane localization is constrained by interactions with Contactin and
Neuroglian (Faivre-Sarrailh et al.,
2004; Laval et al.,
2008), as well as with cytoskeleton-associated proteins important
for membrane localization (Laval et al.,
2008; Ward et al.,
1998b; Wu et al.,
2007). By contrast, the localization of Nrx-IV in neurons appears
relatively fluid and dispersed, only accumulating at high levels when in
contact with a Wrapper+ membrane. It remains possible that
once Wrapper and Nrx-IV bind, additional proteins might bind to Nrx-IV to
stabilize its membrane localization. These interactions could further regulate
the dynamics of MG-neuron interactions.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/7/1147/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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2 cells, averaged between two experiments. Error
bars show s.d. (D,E) Drosophila S2 cells were
transiently transfected with (D) Nrx-IV alone and (E)
wrapper alone. (F) Nrx-IV- and
wrapper-transfected cells were mixed together. (G-I) S2 cells
were transiently transfected with wrapper and immunostained for
Wrapper and Nrx-IV. Wrapper- cells are outlined (dotted lines).
Scale bar: 5 µm