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First published online 22 November 2006
doi: 10.1242/dev.02653
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1 Department of Neuroscience, University of Pennsylvania School of Medicine, 421
Curie Boulevard, Philadelphia, PA 19104, USA.
2 Program in Cell and Developmental Biology, and Department of Biological
Sciences, University of Illinois Chicago, Chicago, IL 60607, USA.
* Author for correspondence (e-mail: gbashaw{at}mail.med.upenn.edu)
Accepted 19 September 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and ß-Spectrin are major components of a submembrane
cytoskeletal network connecting actin filaments to integral plasma membrane
proteins. Besides its structural role in red blood cells, the Spectrin network
is thought to function in non-erythroid cells during protein targeting and
membrane domain formation. Here, we demonstrate that ß-Spectrin is
required in neurons for proper midline axon guidance in the
Drosophila embryonic CNS. In ß-spectrin mutants many
axons inappropriately cross the CNS midline, suggesting a role for
ß-Spectrin in midline repulsion. Surprisingly, neither the
Ankyrin-binding nor the pleckstrin homology (PH) domains of ß-Spectrin
are required for accurate guidance decisions. -Spectrin is dependent
upon ß-Spectrin for its normal subcellular localization and/or
maintenance, whereas -spectrin mutants exhibit a
redistribution of ß-Spectrin to the axon scaffold.
ß-spectrin mutants show specific dose-dependent genetic
interactions with the midline repellent slit and its neuronal
receptor roundabout (robo), but not with other guidance
molecules. The results suggest that ß-Spectrin contributes to midline
repulsion through the regulation of Slit-Robo pathway components. We propose
that the Spectrin network is playing a role independently of Ankyrin in the
establishment and/or maintenance of specialized membrane domains containing
guidance molecules that ensure the fidelity of axon repulsion at the
midline.
Key words: Axon guidance, Midline, Repulsion, Roundabout, Slit, Spectrin, Ankyrin, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Slit ligands and Robo receptors play conserved roles in
regulating midline axon crossing in both invertebrate and vertebrate nervous
systems. In the Drosophila embryonic CNS, ipsilateral neurons
(neurons that do not cross the midline) express the Robo receptor on their
surface, which senses Slit to prevent midline crossing
(Kidd et al., 1999;
Kidd et al., 1998). By
contrast, commissural neurons can overcome Slit repulsion because expression
of Robo on their surface is prevented prior to midline crossing by the
Commissureless (Comm) protein (Keleman et
al., 2002). Thus, the specific receptors that a growth cone
expresses as it encounters the midline greatly influences its guidance
decision. The question of how guidance receptors and their downstream
effectors are targeted to and distributed within functional domains of the
growth cone plasma membrane remains key to the understanding of the mechanisms
of axon path finding.
The Spectrin molecule, a long rod-shaped heterotetramer consisting of two
and two ß subunits, is the defining element of a ubiquitous
sub-membrane cytoskeletal network in nearly all metazoan cells
(Bennett and Baines, 2001).
Most ß-Spectrin isoforms contain an actin-binding domain at their N
termini, an Ankyrin-binding domain, and a pleckstrin homology (PH) domain near
their C termini. The PH domain of ß-Spectrin interacts with the plasma
membrane in transfected COS cells (Wang et
al., 1996). -Spectrin contains an SH3 domain and two
EF-hand motifs (Bennett and Baines,
2001).
In red blood cells, the Spectrin network was originally shown to have a
role in supporting cell shape (Bennett and
Chen, 2001); however, more recently it has been shown to
participate in the formation of specialized membrane sub-domains
(Bennett and Chen, 2001).
Through its binding partner Ankyrin, the Spectrin network links many integral
membrane proteins to the actin cytoskeleton. Genetic studies have revealed
that many membrane proteins are mislocalized in the absence of Spectrin or
Ankyrin (Dubreuil et al.,
2000; Jenkins and Bennett,
2001; Komada and Soriano,
2002; Zhou et al.,
1998). In addition, mutations in the vertebrate ß-Spectrin
and Ankyrin genes have been linked to various diseases, such as hereditary
hemolytic anemias and spinocerebellar ataxia in humans, and auditory and motor
neuropathies in mice (Ikeda et al.,
2006; Mohler and Bennett,
2005; Parkinson et al.,
2001). It has been proposed that Ankyrin and ß-Spectrin are
interdependent, and mutually stabilize the formation of polarized domains at
axon initial segments and nodes of Ranvier
(Komada and Soriano, 2002).
Yet the relationship between Ankyrin and ß-Spectrin, and the role that
each plays in coordinately regulating microdomain assembly, is not well
understood.
In Drosophila, ß-spectrin mutants die before larval
hatching and loss of ß-Spectrin leads to a failure of Na,K ATPase
accumulation at the basolateral domain of midgut epithelial cells
(Dubreuil et al., 2000). More
recently, studies using RNA interference (RNAi) techniques have demonstrated a
role for ß-Spectrin during nervous system development. Specifically, the
ß-Spectrin network was shown to be required pre-synaptically to maintain
cell adhesion molecule (CAM) organization and neuromuscular junction stability
(Pielage et al., 2005).
In a genetic screen for additional genes involved in midline axon guidance
in the Drosophila CNS, we identified three ß-spectrin
alleles. ß-spectrin mutant embryos display fused anterior and
posterior commissures, and ectopic Fas2-positive neurons crossing the midline,
suggesting a role for Spectrin in midline repulsion. Likewise,
-spectrin mutants exhibit very mild defects in midline
repulsion, supporting the idea that it too has a minor role in regulating
guidance. Additionally, in -spectrin mutants, ß-Spectrin
is preferentially distributed to CNS axons, suggesting that -Spectrin
normally limits axonal accumulation of ß-Spectrin. Genetic rescue
experiments demonstrate that ß-Spectrin functions in neurons and that the
Ankyrin-binding domain is not required for guidance. This was surprising as
Ankyrin is thought to be intimately associated with Spectrin in most cases.
Furthermore, ß-spectrin mutants demonstrate dose-dependent
genetic interactions with the repulsive ligand slit and its receptor
robo (but not with other guidance molecules), suggesting that they
may be involved in the same pathway. Similar genetic interactions are observed
within a small subpopulation of ipsilateral neurons, the apterous (Ap)
neurons. Defects in the Ap neurons appear later in development, suggesting a
role for ß-spectrin in the maintenance of proper connectivity.
Our data support a model in which the Spectrin network is important for the
sorting and/or localization of molecules that contribute to and maintain
midline repulsion.
Hülsmeier et al. (Hülsmeier
et al., 2007) have reported that ß-spectrin is the
gene disrupted in the karussel mutant; karussel was
previously isolated in a large-scale genetic screen for defects in axon
guidance at the CNS midline (Hummel et
al., 1999b). Our results, together with this complementary study,
strongly support an important role for ß-spectrin in the
regulation of connectivity in the embryonic CNS.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Specrg41/Tm3UbxßGal; (7)
Speclm88/Tm3UbxßGal; (8)
Speclm32/Tm3UbxßGal; (9) slit2,
roboGA285/CyOWgßgal; (10) slit2,
roboGA285/CyOWgßgal; elavGAL4; (11) slit2, aptGAL4,
UAS-TauMycGFP/CyOTubulinGal80; (12) aptGAL4, UAS-TauMycGFP/CyOTubulinGal80;
(13) roboGA285, aptGAL4/CyOWgßgal; (14)
roboGA285/CyOWgßgal; UAS-TauGFP; (15) ywiso3; (16)
elavGAL4 (III); and (17) single-mindedGAL4 (simGAL4).
Genetic screen
To identify genes involved in midline guidance, we screened for defects in
the eagle neurons, a subset of commissural neurons that require
frazzled for midline attraction (D.S.G. and G.J.B., unpublished)
using a reporter stock: fra, UAS-TauMycGFP/CyTubulinGal80; eagleGAL4.
Reporter males were crossed to a collection of lethal p-element lines on the X
chromosome. Embryos were fixed and stained using a `large-scale collection'
technique modified from Hummel et al.
(Hummel et al., 1997). General
axon guidance defects were identified using anti-BP102 and subset guidance
errors were identified using anti-GFP.
Tissue-specific rescue
ßspecem6/FM7actinßgal;
UASßSpectrin-Myc flies were generated and crossed to
elavGAL4 or simGAL4 males.
Spec enhancement
ßSpechypo/FM7actinßgal;
CxD/TM3Ubxßgal virgins were crossed to
Speclm88 or
rg41/TM3Ubxßgal males. Non-FM7, non-CxD
virgins (ßSpec/+; Spec/TM3Ubxßgal)
were crossed back to Spec/TM3Ubxßgal males.
Hemizygous ß-spectrin and homozygous -spectrin
mutant embryos were identified by the absence of staining for Sex Lethal
(Sxl), ß-Spectrin or ß-gal.
Structure/function study
Male flies carrying ubiquitously expressed wild-type (KW3A) or mutant
transgenes (ßSpec
ank or ßSpecPH) were crossed to
ßSpecem6/FM7actinßgal virgins. Embryos negative for Sxl
and ß-gal but positive for Myc were scored for rescue using an
anti-1D4/Fas2 antibody.
Immunohistochemistry
The following primary antibodies were used: (1) mouse anti-1D4/Fas2
[Developmental Studies Hybridoma Bank (DSHB), 1:100]; (2) mouse anti-BP102
(DSHB, 1:100); (3) rabbit anti-Myc (Sigma-Aldrich, 1:500); (4) rabbit
anti-ß-Spectrin [337 (Byers et al.,
1989; Dubreuil et al.,
2000)]; (5) mouse anti--Spectrin (DSHB, 3A9, 1:25); (6)
mouse anti-Sex Lethal (DSHB, M18, 1:1000); (7) mouse anti-ß-gal (DSHB,
40-1a, 1:250); (8) Rb-anti-GFP (Molecular Probes, 1:500); (9) mouse anti-Robo
(DSHB, 1:50). The following secondary antibodies were used: AlexaFluor488 goat
anti-Rb (Molecular Probes, 1:500) and Cy3-conjugated goat anti-mouse (Jackson
Laboratories, 1:1000). Stacks of images were obtained using a Leica DMIRE2
confocal and a 63x oil immersion objective. Stacks were generated using
NIH ImageJ software.
Molecular biology
We confirmed the online p-element insert sequence annotation
(http://flybase.bio.indiana.edu/)
by recovering and sequencing the genomic DNA flanking the p-elements using
standard protocols and primers (Huang et
al., 2000). Although the p-element insertion sites associated with
alleles G0108 and G0074 are only approximately 60
nucleotides apart from each other (Fig.
1A), G0108 embryos display a much more severe defect than
do G0074 embryos.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Hummel et al., 1999b;
Seeger, 1994). In an attempt
to identify additional axon guidance molecules, we screened for global axon
guidance defects and also more specifically for axon guidance errors within a
subset of commissural neurons, the eagle neurons, labeled with GFP (see
Materials and methods). We screened a collection of lethal p-elements on the X
chromosome and identified a p-element line (0011819, allele G0108),
in which hemizygous males exhibit a compressed axon scaffold containing fused
anterior and posterior commissures. Public database
(http://flybase.bio.indiana.edu)
annotation indicates that the p-element disrupts the ß-spectrin
locus (Fig. 1A). There were two
additional ß-spectrin alleles
(Fig. 1A) contained in the
p-element collection (line 11799, allele G0074; and line 11922,
allele G0198), both of which display phenotypes similar to the
originally identified allele, although less severe
(Fig. 1I). A strong
loss-of-function (lof) ß-spectrin allele, em6, and an
allele that creates a truncated ß-Spectrin protein, em21
(Dubreuil et al., 2000), also
have similar phenotypes (Fig.
1I). In addition, mutant p-element embryos show reduced levels of
ß-Spectrin protein (see Fig. S1 in the supplementary material). Taken
together, these data indicate that the p-elements that cause axonal defects
disrupt ß-spectrin gene function.
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),
and confirmed that the midline glia are present in the appropriate number and
positions (Fig. 1L). The
midline glia in ß-spectrin mutants also express Slit, suggesting
they produce appropriate repulsive guidance cues (data not shown).
Often, when midline repulsion is compromised, the most medial Fas2-positive
longitudinal fascicle inappropriately crosses the midline and continues to
extend on the opposite side (Kidd et al.,
1998). Therefore, we investigated whether ß-spectrin
mutants had inappropriate Fas2-positive bundles crossing the midline. Similar
to wild type, ß-spectrin heterozygous embryos have no
inappropriate crossovers (Fig.
1E,I). By contrast, ß-spectrin hemizygous mutant
embryos display ectopic Fas2-positive neurons crossing the midline
(Fig. 1F,I). em6, em21
and the p-element allele G0108 show the strongest crossing defects,
whereas the presumptive hypomorphic p-element alleles (G0198 and
G0074) have less severe defects
(Fig. 1I). Additionally, when
compared with wild type, ß-spectrin mutant embryos show a
reduced distance between opposing Fas2-positive medial longitudinal bundles
(Fig. 1E,F), and the most
lateral Fas2-positive bundles of axons appear to stall and fail to extend
longitudinally (Fig. 1F,
arrowheads). In C. elegans, mutations in ß-spectrin
(unc-70/bgs-1) also appear to affect neuronal function and axon
outgrowth (Hammarlund et al.,
2000; Moorthy et al.,
2000), which suggests that Drosophila ß-Spectrin may
be playing a conserved role during the outgrowth of certain classes of
axon.
ß-Spectrin is required in neurons for midline axon guidance
To investigate where ß-Spectrin functions during midline axon
guidance, we examined ß-Spectrin protein expression in wild-type and
ß-spectrin mutant embryos using an antibody that specifically
recognizes full-length ß-Spectrin
(Byers et al., 1989;
Dubreuil et al., 2000).
Wild-type ß-Spectrin localizes weakly to the axon scaffold and is
targeted to the plasma membrane surrounding every cell within the midline
neuropil (Fig. 1J).
em6 embryos have greatly reduced levels of ß-Spectrin protein
(Fig. 1K), yet we cannot rule
out the possibility that trace levels may be maternally loaded. Strong and
weak loss-of-function p-element alleles exhibit reduced levels and
mislocalization (including lack of complete uniform cell body plasma membrane
staining) of ß-Spectrin protein (see Fig. S1 in the supplementary
material).
The ubiquitous expression pattern of ß-Spectrin did not give any insight into where ß-Spectrin is required for its midline guidance function. Therefore, to investigate this, we tested two candidate cell types - neurons and midline glia - by expressing wild-type ß-Spectrin protein in both cell types using the Gal4/UAS system in ß-spectrin mutants. We found that the mutant phenotypes can be rescued by expressing wild-type UAS-ß-Spectrin in all postmitotic neurons using elavGAL4 (Fig. 1D,G). By contrast, expressing ß-Spectrin in midline glia under the control of simGAL4 does not rescue the ß-spectrin phenotype (Fig. 1H). Importantly, overexpression of ß-Spectrin throughout the CNS in a wild-type background using elavGAL4 does not result in any obvious defects (data not shown). These data demonstrate that ß-Spectrin is required in neurons but not in other cell types at the midline, and support the idea that ß-Spectrin contributes to axon repulsion.
-Spectrin plays a minor role during midline axon repulsion
Canonically, - and ß-Spectrin function together as a
heterotetramer that ubiquitously associates with the plasma membrane
(Bennett and Baines, 2001;
De Matteis and Morrow, 1998).
Therefore, we investigated whether -Spectrin, like ß-Spectrin, has
a role during midline axon guidance. In contrast to what is observed in
ß-spectrin mutant embryos, -spectrin mutants
display only very mild defects in axon guidance. Specifically, these errors
are seen just in certain hetero- and homoallelic combinations
(Fig. 2A,C,F). When both copies
of -spectrin are removed in a hypomorphic hemizygous
ß-spectrin background, we see an increase in the number of
Fas2-positive axon bundles ectopically crossing the midline
(Fig. 2D-F). This genetic
interaction supports the idea that -spectrin modestly
contributes to axon guidance. However, ß-Spectrin appears to be playing a
much larger role during this developmental process. Hulsmeier et al.
independently identified additional -spectrin alleles that do
not display guidance defects
(Hülsmeier et al., 2007).
These data further support the idea that -spectrin plays only
a minor role during midline guidance.
- and ß-Spectrin depend on each other for proper localization
To investigate the relationship between - and ß-Spectrin, we
examined their expression patterns in wild-type and single mutant backgrounds.
Wild-type - and ß-Spectrin colocalize at the plasma membrane
around every cell in the CNS and are weakly expressed on axon tracks
(Fig. 3A-C). As noted above,
ß-spectrin (em6) mutant embryos exhibit severely
reduced levels of ß-Spectrin protein
(Fig. 3D). Similarly, when
image parameters are kept constant, -Spectrin protein levels are also
greatly reduced in ß-spectrin mutants. However, the Photo
Multiplier Tube (PMT) gain can be increased to detect low levels of
-Spectrin at the plasma membrane surrounding the cell bodies of the
CNS, although the protein looks slightly more diffuse than in wild type
(Fig. 3E, starred arrowheads).
The fact that levels of -Spectrin are dramatically reduced in
ß-spectrin mutant embryos suggests that localization and/or
maintenance of -Spectrin depends on ß-Spectrin. However, the fact
that we still see some -Spectrin would suggest that there may be a
minimal supply of maternally deposited ß-Spectrin still present (although
barely detectable by our antibody) in the ß-spectrin mutant
embryos that is able to localize -Spectrin. We also cannot rule out the
alternative possibility that there is a small pool of -Spectrin that
can localize independently of ß-Spectrin. When we stain embryos carrying
the hypomorphic allele (G0108) with anti-ß-Spectrin and
anti--Spectrin, we see reduced levels and mislocalization of both
proteins. Intriguingly, -Spectrin still colocalizes with
ß-Spectrin (see Fig. S1 in the supplementary material) even though the
overall expression pattern is highly disrupted. This is consistent with the
hypothesis that ß-Spectrin contributes to the localization and/or
maintenance of -Spectrin.
As expected, -spectrin mutants have reduced levels of
-Spectrin protein (Fig.
3H). The residual amount of -Spectrin protein present in
the embryo appears to still localize properly to the cell body plasma
membrane, but not to axons (Fig.
3H, arrows), and is likely to be maternally contributed. In marked
contrast to the clear dependence of -Spectrin expression and/or
stability on wild-type ß-Spectrin, - spectrin
mutants exhibit a noticeable redistribution of ß-Spectrin to the axon
scaffold (compare Fig. 3A with
3G). Thus, it appears that -Spectrin is normally required
to keep axonal levels of ß-Spectrin low.
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).
Therefore, we were interested in testing whether there was a similar
relationship between Ankyrin and ß-Spectrin in Drosophila.
Currently, dank1 (Drosophila ankyrin-1; ankyrin -
FlyBase) mutants are not available but their interaction can be studied by
using a mutant ß-Spectrin transgene in which Ankyrin-binding activity has
been removed (Das et al.,
2006). Specifically, this ßSpecank transgene cannot
rescue the localization defects of Dank1::EGFP in copper cells of
ß-spectrin mutants, suggesting that Ank1-binding is indeed
disrupted. Surprisingly, when expressed ubiquitously throughout the embryo,
ßSpecank rescues the axon guidance defects as efficiently as does
wild-type ß-Spectrin (Fig.
4C,D,F). Because we know that ß-Spectrin is required in
neurons (Fig. 1), we attribute
this rescue to its neuronal function. These data suggest that the role of
ß-Spectrin during midline axon guidance in Drosophila is
independent of Dank1.
Interestingly, Ankyrin-independent membrane binding sites for
ß-Spectrin were identified in binding studies with NaOH-stripped
membranes from rat brain (Davis and
Bennett, 1994), and a reporter consisting of the PH domain of
vertebrate ßI-Spectrin fused to GFP binds IP3 and mediates targeting to
the plasma membrane in COS cells (Wang et
al., 1996). Therefore, to address whether the Ankyrin-binding or
PH domains are required for precise localization of ß-Spectrin in
Drosophila neurons, we used the Myc epitope tag to monitor transgene
expression. As expected, ubiquitously expressed full-length ß-Spectrin
localizes to axons and the plasma membrane of all cells in the neuropil
(Fig. 4H,K). Similarly,
ßSpecank localizes with the same pattern as the full-length
protein (Fig. 4I,L). By
contrast, ßSpecPH does not localize to the plasma membrane of cell
bodies and the overall level of expression is reduced when compared with the
other transgenes (Fig. 4J,M).
However, we still observe some axonal staining
(Fig. 4J). Despite disrupted
cell body plasma membrane staining, ßSpecPH completely rescues the
midline guidance defects observed in ß-spectrin mutant embryos
(Fig. 4E), suggesting that the
PH domain is not required for ß-Spectrin to carry out its role in
guidance. Together, these data suggest that the PH domain, but not the
Ankyrin-binding domain, is required for proper membrane localization in the
cell body. However, cell body plasma membrane localization does not appear to
be required for function, as ßSpecPH can still rescue the guidance
phenotype despite partially defective distribution. This is consistent with
the idea that a sub-population of axonally localized ß-Spectrin is
important during growth cone migration, and this localization is independent
of the PH and Dank1-binding domains. However, we do acknowledge that these
gain-of-function experiments may not reflect the true localization of
endogenous proteins with similar deletions. For example, in our experiments,
overexpressed full-length ß-Spectrin appears more concentrated on axons
than does endogenous protein (compare Fig.
1J with Fig.
4H).
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In addition to defects in Fas2-positive neurons, we also see ß-spectrin mutant phenotypes and genetic interactions with the Slit-Robo pathway within a small subpopulation of ipsilateral neurons, the apterous (Ap) neurons. In wild-type embryos, the Ap neurons approach the midline but never cross (Fig. 7A). In both slit/+ heterozygous and ß-spectrin (em6) hemizygous mutant embryos, we observe mild midline errors (`thick crosses') when Ap neurons are labeled with Tau-Myc-GFP (Fig. 7B,C,H). Both null and hypomorphic mutations in ß-spectrin enhance the slit/+ heterozygous phenotype (Fig. 7D,H). In contrast to pan-neural expression, which completely rescues the mutant ß-spectrin guidance defects, the Ap neuron defect cannot be rescued by expressing full-length ß-Spectrin in the Ap neurons (Fig. 7E,H). These data suggest a non-autonomous function for Spectrin for some classes of neuron and support the idea that the overall defective patterning of the entire CNS contributes to defects seen in the Ap neurons. robo mutants also display ectopic Ap neurons crossing the midline (Fig. 7F); however, this defect can be rescued by expressing full-length Robo in these neurons, demonstrating that Robo functions cell autonomously (Fig. 7G).
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Similar to what we have observed for the early behavior of the pCC and dMP2 axons, axons of the Ap neurons initially make the correct steering decisions in ß-spectrin mutant embryos. Specifically, projection toward the midline is normal in early stage 14 embryos and axons make correct anterior ipsilateral turns at late stage 14 to early stage 15 (Fig. 8G,H), similar to wild type (Fig. 8A-C). However midline-crossing defects are observed by late stage 16 (Fig. 8I). By contrast, robo mutants exhibit Ap neuron crossing defects during their initial extension toward the midline and throughout development (Fig. 8D-F). In addition, although ß-spectrin mutants that are also heterozygous for slit show considerably stronger guidance and crossing defects, these defects only arise at later stages of CNS development (Fig. 8K,L). These observations suggest (1) that Slit-Robo repulsion is required not only for initial axon guidance, but also to maintain connections on the correct slide of the midline, and (2) that ß-spectrin contributes to this later maintenance function.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PH or ßSpecAnk mutant proteins are overexpressed.
These overexpression experiments suggest that ß-Spectrin may localize to
axons independently of the Ankyrin-binding and PH domains, and that axonal
ß-Spectrin is sufficient for growth cone migration. However, it is
important to note that because these observations are based on overexpression
studies, they may not completely reflect the localization of endogenous
proteins with similar deletions. Finally, analysis of progressive
developmental stages in ß-Spectrin mutants suggests that, for at
least some neurons, ß-Spectrin is not required for the initial
pathfinding of axons, but rather appears to be important for maintaining
appropriate connectivity. Together, our findings support a role of the
Spectrin network in contributing to the fidelity of axon repulsion at the
Drosophila midline.
The role of ß-Spectrin in midline repulsion during growth cone migration
The midline guidance defects we observe in embryos stained with BP102 and
Fas2 antibodies suggest that ß-Spectrin normally contributes to axonal
migration and more specifically to axon repulsion. Considering that
ß-Spectrin has been shown to modulate the behavior of interacting
membrane proteins within sub-membrane microdomains, and given that we observe
specific dose-dependent genetic interactions with the Slit-Robo pathway, our
data support the idea that the Spectrin cytoskeleton modulates the behavior of
molecules that contribute to Robo repulsion. Many important signaling
molecules must be coordinated downstream of guidance receptors so that
navigating growth cones make appropriate decisions. For example, Dock, Pak and
Rac contribute to midline repulsion by forming a complex with the Robo
receptor upon Slit stimulation (Fan et
al., 2003). Similarly, Drosophila Ena, a member of a
protein family implicated in actin cytoskeleton regulation, functions
cooperatively with Robo at a level downstream of the receptor
(Bashaw et al., 2000).
Additionally, the microtubule-associated protein CLASP (Chb - FlyBase) and the
Abelson tyrosine kinase are required to induce restricted cytoskeletal events
at the leading edge of growth cones (Lee
et al., 2004). Along these lines, ß-Spectrin may affect the
ability of comparable proteins to signal effectively in the Slit-Robo pathway.
Clearly, the Spectrin network cannot account for all Robo function, as many
ipsilateral CNS neurons are still guided properly in ß-spectrin
mutants. Mutations in ß-spectrin also enhance a robo1
null loss-of-function mutation (data not shown), suggesting that the Spectrin
network probably plays an additional role in repulsion outside of Robo1
signaling. It is important to note that our data do not rule out the
alternative possibility that ß-spectrin mutations may have
general effects on growth cone migration. If so, then the specific
interactions seen with slit and robo might reflect the fact
that the Slit-Robo pathway is more susceptible to subtle perturbations than
are the other guidance pathways tested.
ß-Spectrin and the maintenance of axon connections
Our analysis of the effect of ß-Spectrin mutations on the
guidance of Ap neurons suggests that ß-Spectrin is required not
for the initial pathfinding of these axons, but rather for maintaining
correctly established connections. Moreover, our genetic analysis suggests
that in this context the Slit and Robo system also contributes to this
maintenance function. Our observations support the idea that some level of
Slit/Robo repulsion is required continuously to keep ipsilateral axons on
their own side of the midline. A more dramatic example of pathway maintenance
has been described in C. elegans, where mutations in the Zig genes
lead to a `flipping' of axon pathways over the midline
(Aurelio et al., 2003;
Aurelio et al., 2002).
Additionally, in Drosophila, it has been shown that ß-Spectrin
is essential for synapse stabilization
(Pielage et al., 2005). It
will be interesting in the future to determine whether the maintenance
function of slit, robo and ß-Spectrin represents a
repulsive mechanism distinct from the mechanism operating during pathway
formation.
Relationships between - and ß-Spectrin in the Drosophila CNS
Our work demonstrates that in the absence of ß-Spectrin the stability
of -Spectrin is decreased. Thus, it appears that hetero-tetramer
formation is required to maintain proper levels of -Spectrin in the
nervous system. By contrast, cell body plasma membrane localization of
ß-Spectrin is unaffected in -spectrin mutants. Consistent
with this result, it has been shown that ß-Spectrin accumulates
independently of -Spectrin in Drosophila larvae
(Dubreuil and Yu, 1994). This
suggests that ß-Spectrin recruitment to and stability at the cell body
plasma membrane are independent of -Spectrin, and supports the idea of
an -Spectrin-independent role for ß-Spectrin in neurons (see
below).
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- and ß-Spectrin function together as a hetero-tetramer, why
then do we observe only mild axon guidance defects in
-spectrin single mutants? One explanation is that perhaps
-Spectrin has a higher maternal component that is able to compensate
for a lack of zygotic expression. Indeed, -spectrin mutants
survive to a later developmental stage than do ß-spectrin
mutants. However, -spectrin mutants also exhibit increased
levels of axonally localized ß-Spectrin (see below). Therefore, an
alternative hypothesis could be that the preferential distribution of
ß-Spectrin to axons somehow compensates for a reduction of
-Spectrin, allowing neurons to make precise steering decisions. Lastly,
and perhaps most interesting, ß-Spectrin may function independently of
-Spectrin in neurons. This idea seems plausible given that
ß-Spectrin remains properly localized to the cell body plasma membrane in
-spectrin mutants.
As noted above, -spectrin mutant embryos show an increase
in the levels of axonal ß-Spectrin, suggesting that -Spectrin
regulates the accumulation of ß-Spectrin in axons. What does this shift
in ß-Spectrin distribution mean for the neuron? What are the signals that
target ß-Spectrin to axons and why does the localization change in
-spectrin mutants? One possibility is that the SH3 domain of
-Spectrin targets ß-Spectrin to the cell body plasma membrane via
a direct interaction with another protein. Another possibility is that, in the
absence of -Spectrin, ß-Spectrin binds other axonally localized
proteins to a greater degree, thus shifting the overall distribution.
Domain requirements for ß-Spectrin axon guidance function
It was originally thought that Ankyrin-G is assembled upstream of
ßIV-Spectrin at axon initial segments
(Jenkins and Bennett, 2001);
however, later reports suggested that both Ankyrin-G and ßIV-Spectrin are
required for the localization and stability of one another, as well as for the
stability of VGSCs at axon initial segments and nodes of Ranvier
(Komada and Soriano, 2002).
Yet, in neonatal cardiomyocytes, Ankyrin-B is required for the proper
distribution and levels of ßII-Spectrin, and an Ankyrin-B protein lacking
the Spectrin-binding domain still localizes properly. These data suggest that
Ankyrin-B localization is independent of ßII-Spectrin. We sought to test
the functional importance of the protein domains of ß-Spectrin during
axon guidance, and the order of assembly of ß-Spectrin and Ankyrin in
Drosophila neurons.
|
ank) remains
properly localized to cell bodies and axons. Importantly, a recent study has
shown that the same ßSpecank protein used in this study correctly
accumulates at the plasma membrane of copper cells yet fails to accurately
target an Ank-GFP fusion protein in epithelial cells
(Das et al., 2006), suggesting
that Ankyrin localization depends on ß-Spectrin but not vice versa. In
addition, ßSpecank rescues the guidance errors seen in
ß-spectrin mutants. Taken together, our data suggest that
Ankyrin-binding is not essential for ß-Spectrin localization and guidance
function in neurons, and that Drosophila ß-Spectrin may be
assembled upstream of Ankyrin.
It is important to note that Drosophila has another Ankyrin gene,
dank2 (ank2 - FlyBase), which is expressed specifically in
neurons (Hortsch et al.,
2002). Therefore, perhaps accurate ß-Spectrin targeting
occurs via a direct interaction with Dank2. Keeping in mind that
ßSpecank is missing the conserved Ankyrin1-binding site, this
logic would imply that ß-Spectrin associates with Dank2 using a different
binding site than that used for Dank1. There does appear to be an intimate
relationship between Dank2 and ß-Spectrin, as Dank2 is mislocalized and
levels are reduced in ß-spectrin mutants (D.S.G. and G.J.B.,
unpublished). Future experiments will help to establish whether Dank2 can bind
ß-Spectrin at a location different than that used by Dank1. If the mutant
ßSpecank does indeed bind to Dank2, it will be interesting to
determine the functional significance of this interaction.
We also demonstrated that, although the PH domain of Drosophila ß-Spectrin is not necessary for axonal localization and/or guidance function, it is required for appropriate localization to the cell body plasma membrane. From our experiments, it appears that axonally localized ß-Spectrin is important for making accurate guidance decisions. In the future, it will be important to determine the region of ß-Spectrin that is essential for axonal targeting and whether this domain is important for growth cone migration.
Implications for cell adhesion molecules in midline axon guidance
At nodes of Ranvier and axon initial segments, there is an intimate
relationship between the Spectrin network, Ankyrin and CAMs
(Lambert et al., 1997). For
example, in Purkinje and granule cells, Ankyrin-G localization precedes that
of neural CAMs, and is required for proper CAM cluster assembly at axon
initial segments (Jenkins and Bennett,
2001; Zhou et al.,
1998). Other studies of CAMs have revealed that they can commonly
act as contactmediated attractive and repulsive signals for growing axons
(Doherty and Walsh, 1994;
Tanaka and Sabry, 1995). In
Drosophila, pre-synaptically targeted ß-spectrin RNAi
disrupts the stability and organization of CAMs such as Fas2 and Neuroglian
(Pielage et al., 2005), and
neuroglian mutants exhibit defects in motoneuron pathfinding
(Hall and Bieber, 1997).
Furthermore, neurotactin mutant embryos exhibit midline axon guidance
errors that appear to be similar to those observed in ß-spectrin
mutants, including ectopic midline crossing and longitudinal breaks
(Speicher et al., 1998).
Future studies should investigate whether there are functional links between
CAMs and the Spectrin network in the context of midline axon guidance.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/2/273/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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