1 Unit on Neuronal Connectivity, Laboratory of Gene Regulation and Development,
National Institute of Child Health and Human Development, National Institutes
of Health, Bethesda, MD 20892, USA
2 Department of Cell and Structure Biology, University of Illinois, Urbana, IL
61801, USA
Author for correspondence (e-mail:
leechih{at}mail.nih.gov)
Accepted 22 December 2004
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SUMMARY |
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Key words: Drosophila, N-cadherin, R7
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Introduction |
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The Drosophila visual system contains projections from the eye to
the brain that segregate in specific layers; the targeting of these
projections is genetically hardwired
(Clandinin and Zipursky, 2002;
Tayler and Garrity, 2003
). It
has been used as a model system for studying the development of layer-specific
connections. The Drosophila compound eye consists of approximately
800 ommatidia, each containing three types of photoreceptor neurons (R1-R6,
R7, and R8) (Meinertzhagen and Hanson,
1993
). The R1-R6 neurons respond maximally to the blue/green
spectrum of light and connect to the first optic neuropil (the lamina),
whereas the R7 and R8 are most sensitive to the ultraviolet and blue or green
spectra of light, respectively, and connect to the second optic neuropil (the
medulla) (Salcedo et al.,
1999
). The medulla is subdivided into ten layers (M1-M10) based on
the terminals of the innvervating afferents: the R7 and R8 axons project to
the M6 and M3 layers, respectively, while the lamina neurons (L1-5) relay
R1-R6 input to multiple medulla layers
(Fischbach and Dittrich,
1989
).
Genetic screens based on visual behaviors or histology identified three
surface receptors, the Drosophila N-cadherin (Ncad; CadN - FlyBase),
the receptor tyrosine phosphatases LAR, and PTP69D, that are required for R7
layer selection (Clandinin et al.,
2001; Lee et al.,
2001
; Maurel-Zaffran et al.,
2001
; Newsome et al.,
2000
). N-cadherin and LAR are required cell-autonomously in the R7
neurons while PTP69D might function in R7 or R8 neurons. Removing N-cadherin
or LAR in the R7 neurons results in mutant R7 afferents mistargeting to the
R8-recipient layer, and defects in wavelength-discrimination visual behavior.
LAR is required for R7 afferents to remain in appropriate target layers during
development. The mechanism of action is not known for N-cadherin. The
Drosophila N-cadherin has a larger complex extracellular domain that
mediates homophilic interactions (Iwai et
al., 1997
). It is evolutionarily conserved in worms, insects, and
vertebrates (Broadbent and Pettitt,
2002
; Tanabe et al.,
2004
), and thus may be the most ancient form of classic cadherins.
In this study, we investigated the developmental processes of medulla layer
formation and the role of N-cadherin therein.
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Materials and methods |
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For genetic cell-ablation experiments, hh1
(eye-specific hedgehog allele
(Huang and Kunes, 1996), a
gift from Sam Kunes), sevd2
(Basler and Hafen, 1989
), and
UAS-EGFRDN were used. To block the differentiation of L1-L5
neurons, we incubated the Gcm-Gal4 UAS-EGFRDN embryos at
17°C to suppress Gal4 activity until they reached the third instar stage.
Then, they were moved to a 25°C incubator to allow Gcm-Gal4 to drive the
expression of EGFRDN in the lamina precursors. The fly stocks were
maintained under standard culture conditions at 25°C unless stated
otherwise.
The GMR-Flp/MARCM system for generating mosaic R7 neurons has been
described (Lee et al., 2001).
For analyzing single wild-type or mutant R7s at the 17% and 35% APF, we
included the Elav-Gal4 driver in the genetic scheme. Fly stocks that were used
for these experiments are as follows: (1) GMR-Flp; FRT40; (2)
GMR-Flp; Ncad405 FRT40/CyO, ubiP-GFP; (3) GMR-Flp;
NcadB11 FRT40/CyO, ubiP-GFP; (4) GMR-Flp;
NcadM19 FRT40/CyO, ubiP-GFP; (5) GMR-Flp;
LAR2127 FRT40/CyO, ubiP-GFP; (6) GMR-Flp;
Ncad405 LAR2127 FRT40/CyO, ubiP-GFP; (7)
Elav-Gal4c155, UAS-mCD8-GFP; tubP-Gal80 FRT40.
For MARCM rescue experiments, the following stocks were used: (1) GMR-Flp, UAS-Ncad7b-13a-18a; Ncad405 FRT40/CyO, ubiP-GFP; (2) GMR-Flp; Ncad405 FRT40/CyO, ubiP-GFP; UAS-Ncad7a-13a-18a/TM6b; (3) GMR-Flp; Ncad405 FRT40/CyO, ubiP-GFP; UAS-Ncad7b-13b-18a/TM6b; (4) GMR-Flp; Ncad405 FRT40/CyO, ubiP-GFP; UAS-Ncad7b-13a-18b/TM6b.
The standard MARCM technique (Lee and
Luo, 1999) was used to generate single-cell clones of L1-L5 and R8
neurons or multiple-cell clones of medulla neurons. Two different heat-shock
regimes were used: for generating single-cell lamina clones, 3rd instar lavae
were heat-shocked at 37°C for 10 minutes; for medulla neurons or R8s, 2nd
instar larvae were heat-shocked at 37°C for 40-60 minutes. The following
stocks were used in these experiments: (1) FRT40; (2)
Ncad405 FRT40/CyO, ubiP-GFP; (3) NcadM19
FRT40/CyO, ubiP-GFP; (4) Elav-Gal4c155, UAS-mCD8-GFP,
hs-Flp; tubP-Gal80 FRT40.
Histology
Immunohistochemistry was performed as described previously
(Lee et al., 2001). Confocal
images were acquired using a Zeiss 510 META laser scanning microscope. The
obtained z-stacks of images were deconvolved to remove out-of-focus
light and z-distortion with Huygens Professional software (Scientific
Volume Imaging) running on a 48 processors SGI Origin 2000. The 3D images were
rendered from the restored z-stacks using Imaris software
(Bitplane).
Ncad genomic sequence and transcript analyses:
Blast searching using Bioperl scripts
(Stajich et al., 2002) and
web-based servers was performed to identify potential alternative exons, which
were further examined for appropriate splicing donor and acceptor sites. The
Ncad cDNA corresponding to the 7b-13a-18b isoform
(Iwai et al., 1997
) was used
as query to search against the genomic sequences of Drosophila
melanogaster (Adams et al.,
2000
), Drosophila pseudoobscura, Anopheles gambiae
(Holt et al., 2002
), and
Apis mellifera. In addition to the exons 7b, 13a, and 18a reported
previously (Iwai et al.,
1997
), these analyses identified three potential exons
corresponding to the exons 7a, 13b, and 18b
(Fig. 5).
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Molecular biology
The Ncad isoform 7b-13a-18a and E-cadherin S2 expression vectors and cDNAs
were generous gifts from Tadashi Uemura
(Iwai et al., 1997;
Oda et al., 1994
). The
pRmHa3/Ncad vectors, for expressing different Ncad isoforms in S2 cells, and
the pUAS-Ncad vectors, for transgene rescue experiments, were constructed as
follows. The variable regions corresponding to exons 7a, 13b, 18b were cloned
using RT-PCR and confirmed by sequencing. Ncad isoform cDNAs were
constructed by replacing the variable region in the Ncad 7b-13a-18a
cDNA. The resulting Ncad isoform cDNAs, 7a-13a-18a,
7b-13b-18a, and 7b-13a-18b, were inserted into the S2 expression
vector, pRmHa3, or the P-element vector, pUAST. Transgenic flies were
generated using standard microinjection techniques.
Cell-aggregation assay
For expressing different Ncad isoforms in suspension S2 cells, different
Ncad isoform expression vectors (pRmHa3/Ncad) were cotransfected with a GFP or
Ds-red expression vector (pRmHa3/GFP and pRmHa3/Ds-red). Suspension S2 cells
were a generous gift from James Clement
(Schmucker et al., 2000). Cell
culture and transfection were performed according to the Invitrogen DES and
Qiagen Effectene manuals. CuSO4 was added (0.7 µM) 24 hours
after the transfection to induce the expression of Ncad isoforms and the GFP
or Ds-red marker. The S2 cells were induced for 48 hours and then subjected to
cell-aggregation assay as described previously by Oda
(Oda et al., 1994
) except for
the following modifications. Two populations of S2 cells expressing different
cadherins and markers at the concentration 1.2 x 106 cell/ml
were incubated in 2 ml of the BBS buffer (mM: 10 Hepes, 55 NaCl, 40 KCl, 15
MgSO4, 20 glucose, 50 sucrose) containing 5 mM of CaCl2
in a 35 mm polystyrene dish, and agitated using a gyratory shaker at 100 rpm
for 1.5 hours. The formation of cell aggregates was analyzed under a Zeiss
M2 bio fluorescence microscope with a Zeiss AxioCam digital camera.
Experiments were performed in triplicates.
Western blot
Cell lysis and immunoblotting were performed as described
(Lee et al., 2001). For each
lane, a protein sample equivalent to 200,000 S2 cells was loaded. Rat
monoclonal antibody against the cytoplasmic domain of Ncad proteins
(Iwai et al., 1997
) (a gift
from Tadashi Uemura) was used to detect the Ncad proteins.
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Results |
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The second stage of R7 and R8 target selection starts at approximately 50% APF. Because the R7-specific Gal4 driver, PM181-Gal4, is inactive at this stage, we used a flipase-based system to label late R7 axons (see Materials and methods). We observed that at this stage the R8 growth cones projected past the L2 growth cones to reach the R7-temporary layer, which later became the R8-recipient layer (Fig. 2B,B'). In addition, the R7 axons extended approximately 3-5 µm further into the medulla. During this period, it is possible that as the medulla neuropil continued to develop (see Fig. S2 in the supplementary material), the R-cell growth cones might progress from their temporary layers to their destined layers by processes of active migration and/or passive displacement by the ingrowing medulla processes (or L4-L5 growth cones). By 70% APF, the R7 and R8 growth cones reached their final layers and assumed their adult configurations (Fig. 2D,D'). Thus, layer selection by R7 and R8 axons occurs in two stages. During the first stage, newly differentiating R8 and R7 axons reach their temporary target layers in temporal order according to their time of birth. During the second stage, the R7 and R8 growth cones progress synchronously to their final target layers (Fig. 2H).
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N-cadherin is required for R7 axons to reach and remain in the R7-temporary layer
Using the ultraviolet/visible (UV/Vis) light choice test, we have
previously demonstrated that R7 layer selection requires N-cadherin
(Lee et al., 2001). To
determine the developmental stage during which Ncad functions and to identify
the developmental defects in Ncad mutant R7s, we modified the
single-cell mosaic method previously used for adult phenotypic analysis
(GMR-Flp/MARCM), by including the pan-neuronal driver, Elav-Gal4. This method
allowed us to analyze single wild-type or mutant R7 axons up to 35% APF during
which the Elav driver is active. We observed that at 17% APF, 21%
(n=43) of the Ncad mutant R7 growth cones failed to reach
the R7-temporary layers (Fig.
4B,B'). In addition, many (63%, n=43) Ncad
mutant R7 growth cones exhibited various morphological defects: they failed to
expand fully in the medulla, some expanding prematurely before reaching the
appropriate layer. We next examined the Ncad mutant phenotype at 35%
APF, during which the wild-type R7 and R8 growth cones formed two separate
layers (Fig. 4E,E'). We
observed that 55% (n=26) of the Ncad mutant R7 axons failed
to target to the R7-temporary layer; instead, they terminated at the R8 layer
or the layer between the R7 and R8 layers
(Fig. 4F,F'). Similar
phenotypes and expressivity were observed for three Ncad alleles,
Ncad405, NcadM19, and
Ncadb11 (data not shown). We conclude that at least 24% of
the Ncad mutant R7 growth cones reach the R7-temporary layer at 17%
APF, but retract from the correct layer at 35% APF (results summarized in
Fig. 6E).
|
|
Because Ncad can function as homophilic adhesion molecules in vitro
(Iwai et al., 1997) (see
below), we next examined whether R7 growth cones use Ncad to interact with R8s
or medulla neurons, both of which express Ncad
(Lee et al., 2001
). Using the
MARCM system, we generated Ncad mutant R8 and medulla neuron clones
and assessed layer selection in the corresponding R7 growth cones. Removing
Ncad in single R8 neurons caused R8 growth cones to detach from the
R8-temporary layer (62%, n=13), but had no effect on the
corresponding wild-type R7 axons (0%, n=13; see Fig. S3A,A' in
the supplementary material), indicating that Ncad activity is not required in
R8s for proper R7 layer selection. Similarly, R7 growth cones targeted
correctly to the R7-temporary layer in the presence of small Ncad
mutant medulla neuron clones (0%, 122 lobes examined; see Fig. S3B-D in the
supplementary material). Because of technical difficulty, we were unable to
generate large contiguous medulla neuron patches, and, therefore, could not
rule out the possibility that Ncad would mediate interactions between R7
growth cones and medulla neurons (see Discussion).
The N-cadherin gene undergoes alternative splicing to generate multiple isoforms
The Ncad gene contains three exon modules corresponding to exons
7, 13, and 18, each of which is composed of a pair of highly similar but
distinct exons, designated exons 7a/7b, exons 13a/13b, and exons 18a/18b
(Fig. 5) (see
http://www.flybase.org/.bin/fbidq.html?FBrf0141810).
RT-PCR analysis further revealed that mature Ncad transcripts contain one and
only one of the two alternative exons arising from each exon module (i.e. exon
7a or 7b; 13a or 13b; 18a or 18b), indicating that these exons are used
exclusively (data not shown). All six alternative exons were recovered in the
RNA isolated from developing eye discs. In addition, we uncovered a small
exon, designated as exon 7a', that was found in the exon 7a-containing
Ncad transcripts, but not in those containing exon 7b. Similar Ncad
genomic structure was identified in another member of the Drosophila
family, D. pseudoobscura, the malaria mosquito (Anopheles
gambiae), and, to a lesser extent, the honey bee (Apis
mellifera) (see Fig. S4 in the supplementary material), all of which
diverged from D. melanogaster, approximately 55, 250, and 340 million
years ago, respectively (Gaunt and Miles,
2002; Tamura et al.,
2004
). By combinatorial use of these alternative exons, the
Ncad locus is capable of generating 12 isoforms (encoded by exon 7a,
exon 7a+7a', or exon 7b; exon 13a or 13b; exon 18a or 18b). All the Ncad
alternatively spliced variants share the same domain architecture but have
different sequences in the extracellular or the transmembrane regions.
Expressing single Ncad isoforms is sufficient to rescue R7 targeting defects of Ncad mutants
Because the developing eye discs express multiple Ncad isoforms, we carried
out transgene rescue experiments to address whether multiple Ncad isoforms are
required for R7 targeting. In these experiments, we combined the GMR-Flp/MARCM
system (Lee et al., 2001;
Lee et al., 2000
) with the
UAS-Ncad isoform transgene to express a single type of Ncad isoform in
Ncad mutant R7s. We found that expressing a single Ncad isoform,
including 7a-13a-18a, 7b-13a-18a, 7b-13b-18a, and 7b-13a-18b, substantially,
if not completely, rescues the Ncad phenotypes in R7 axons
(Fig. 6A-E, and data not
shown). We found no significant difference in the abilities of different Ncad
isoform transgenes to rescue the mistargeting or growth cones morphological
defects at either 17% or 35% APF (Fig.
6E).
We next sought to determine whether in the wild-type background, mis- or over-expressing a single Ncad isoform in R7s alters their target specificity. We used the R7-specific Gal4 driver, PM-181-Gal4, to express Ncad isoforms in the wild-type background. We found that expressing any of the isoforms in R7 axons caused modest mistargeting and growth-cone morphological defects (see Fig. S5A',B' in the supplementary material). However, both defects were reduced in the older R7 axons (bracket, see Fig. S5A'',B'' in the supplementary material) and were not observed at the later stage (data not shown). These findings indicate that overexpressing single Ncad isoforms in R7 neurons is insufficient to permanently change the R7 target specificity.
N-cadherin isoforms mediate promiscuous heterophilic interactions
Iwai and colleagues previously reported that the Ncad isoform 7b-13a-18a is
capable of mediating homophilic interaction in a calcium-dependent manner
(Iwai et al., 1997). Because
the alternative isoforms differ in the extracellular and transmembrane
regions, which could potentially affect the binding specificity, we determined
whether the in vitro adhesive activity of Ncad isoforms correlates with their
indiscriminating activity in R7 layer selection. We modified the S2
cell-aggregation assay to test whether Ncad isoforms can mediate heterophilic
interaction with one another and with another Drosophila classic
cadherin, E-cadherin (Oda et al.,
1994
). Two populations of S2 cells that expressed different types
of Ncad isoforms were labeled separately with GFP and Ds-Red and mixed using a
gyratory shaker under constant rotating speed (see Materials and methods for
details). The expression levels of different Ncad isoforms were found to be
similar (see Fig. S6 in the supplementary material). We found that all tested
Ncad isoforms induced mixed-cell aggregates, indicating that they can mediate
heterophilic interactions (Fig.
7B-E). However, the Ncad-expressing S2 cells did not intermix with
the S2 cells expressing E-cadherin; instead, they formed separate cell
aggregates (Fig. 7A). In
summary, the tested Ncad isoforms are capable of mediating type-specific
heterophilic interactions, but they fail to show any detectable isoform
specificity in the in vitro cell-aggregation assays.
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Discussion |
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Using the mutations that delete different afferent subsets or alter R7
connectivity, we defined the mechanism of R7 layer selection. The genetic
cell-ablation results suggest that R8, R7, and L1-L5 afferents target to their
temporary layers independently. In addition, the wild-type R8 axons target
correctly when the neighboring Ncad or LAR mutant R7s
mistarget to the R8-recipient layer (Fig.
4J',K'). Conversely, the removal of Ncad in single R8s
disrupts R8 targeting without affecting the targeting of the neighboring R7s.
Thus, the first stage of medulla layer-selection by R8, R7, and L1-L5
afferents probably involves primarily afferent-target interactions. In
contrast, R1-R6 growth cone sorting to different lamina cartridges involves
both afferent-afferent and afferent-target interactions
(Clandinin and Zipursky, 2000;
Meinertzhagen and Hanson
1993
), even though it required Ncad and LAR
(Lee et al., 2001
;
Clandinin et al., 2001
).
Developmental analyses of single Ncad mutant R7s revealed that
Ncad is required for R7 axons to reach and to remain in the R7-temporary layer
during the first layer-selection stage. On the basis of its homophilic
activity and mutant phenotypes, we previously proposed that Ncad mediates the
interaction between the R7 growth cones and the medulla target neurons. The
medulla contains over 50 different types of neurons
(Fischbach and Dittrich, 1989)
and many express Ncad during development. It is not technologically feasible
at the current stage to remove Ncad activity in all or a large number of
medulla neurons without affecting the pattern of the optic lobe. Thus, even
though we found that removing Ncad in small patches of medulla neurons did not
affect R7 layer selection, we cannot rule out the possibility that multiple
medulla neurons provide redundant N-cadherin-mediated interactions for R7
growth cones in a similar fashion as L1-L5 neurons do for R1-R6 afferents (S.
Prakash and T. Clandinin, personal communication). Alternatively, Ncad might
function as a signaling receptor, rather than as a passive adhesive molecule
in R7s. Recent studies demonstrated that the cytoplasmic domains of vertebrate
classic cadherins could regulate the actin-cytoskeleton via catenins
(Yap and Kovacs, 2003
). The
Drosophila Ncad contains the two conserved cytoplasmic regions that
interact with catenins in vertebrate cadherins. It is conceivable that Ncad
could regulate the actin-cytoskeleton in R7 growth cones in response to
target-derived cues. Furthermore, although Ncad and LAR
share the same adult phenotype, their differential onset of mutant phenotype
and double mutant phenotype suggest that they probably regulate different
aspects of the first R7 layer-selection stage. Ncad is required for R7 growth
cones to initially target to, and remain in the R7-temporary layer throughout
the first target-selection stage, while LAR is only required during the later
phase (Clandinin et al., 2001
;
Maurel-Zaffran et al.,
2001
).
The comparison between the first and second stages of R7 layer selection
reveals a glimpse of the underlying mechanism. First, targeting to the
R7-temporary layer at the first stage appears to be critical for the R7 axons
to reach their final destination. Ncad or LAR mutant R7
axons that mistarget to the R8-temporary layer at the first stage, later
proceed to terminate incorrectly as well at the R8-recipient layer. Second, in
contrast to the initial target selection which follows the axon outgrowth, all
R7 and R8 axons enter the second layer-selection stage at approximately the
same time, regardless of when they arrive at the medulla. Interestingly,
centripetal growth of R1-R6 terminals and synaptogenesis in the lamina
coincide with the second stage of R7 layer-selection
(Meinertzhagen et al., 2000).
It is tempting to speculate that a global signal triggers the initiation of
the second stage.
In this study, we report that the Ncad gene in Drosophila, and probably in other insects, undergoes alternative splicing to generate multiple isoforms. However, the lack of isoform-specificity, revealed by the transgene rescue, overexpression experiments, and heterophilic interaction assays, argues against the hypothesis that the Ncad isoforms constitute an adhesion code to direct targeting specificity. Instead, we favor the idea that Ncad plays a permissive role in R7 layer selection. Nevertheless, the remarkable conservation of the Ncad alternative splicing over 250 million years of evolution suggests an adaptive advantage for Ncad molecular diversity, whose function awaits further investigation.
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Supplementary material |
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ACKNOWLEDGMENTS |
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Footnotes |
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