The Journal of Neuroscience, July 23, 2003, 23(16):6567-6575
Previous Article | Next Article 
N- and C-Terminal Domains of 
-Catenin, Respectively, Are Required to Initiate and Shape Axon Arbors of Retinal Ganglion Cells In Vivo
Tamira M. Elul,1,2
Nikole E. Kimes,1,2
Minoree Kohwi,1 and
Louis F. Reichardt1,2
1Department of Physiology, University of
California San Francisco, and 2Howard Hughes Medical
Institute, San Francisco, California 94143
 |
Abstract
|
|---|
We used deletion mutants to study 
-catenin function in axon
arborization of retinal ganglion cells (RGCs) in live Xenopus laevis
tadpoles. A deletion mutant cat
ARM consists of the N- and
C-terminal domains of wild-type -catenin that contain, respectively,
-catenin and postsynaptic density-95 (PSD-95)/discs large (Dlg)/zona
occludens-1 (ZO-1) (PDZ) binding sites but lacks the central armadillo repeat
region that binds cadherins and other proteins. Expression of ARM in
RGCs of live tadpoles perturbed axon arborization in two distinct ways: some
RGC axons did not form arbors, whereas the remaining RGC axons formed arbors
with abnormally long and tangled branches. Expression of the N- and C-terminal
domains of -catenin separately in RGCs resulted in segregation of these
two phenotypes. The axons of RGCs overexpressing the N-terminal domain of
-catenin developed no or very few branches, whereas axons of RGCs
overexpressing the C-terminal domain of -catenin formed arbors with
long, tangled branches. Additional analysis revealed that the axons of RGCs
that did not form arbors after overexpression of ARM or the N-terminal
domain of -catenin were frequently mistargeted within the tectum. These
results suggest that interactions of the N-terminal domain of -catenin
with -catenin and of the C-terminal domain with PDZ domain-containing
proteins are required, respectively, to initiate and shape axon arbors of RGCs
in vivo.
Key words: -catenin; -catenin; PDZ proteins; lipofection; axon arborization; axon branching; retinal ganglion cells; Xenopus laevis
|
Introduction
|
|---|
In many types of neurons, axons branch or arborize when they reach their
target tissue. Axon arborization is spatiotemporally correlated with
neuron-target interactions (Sakaguchi and
Murphey, 1985
; Yates et al.,
2001) and with synapse formation and function
(Cantallops et al., 2000;
Alsins et al., 2001), but in
contrast to neuron-target interactions and synaptogenesis, we know very little
about the molecules that control axon arborization, with a few notable
exceptions (Cohen-Cory and Fraser,
1995; Wang et al.,
1999; Zou and Cline,
1999).
-catenin participates in several molecular interactions that could
regulate axon arborization. First, -catenin links the cadherin family of
cell adhesion molecules to -catenin and the cytoskeleton, thereby
strengthening adhesion (for review, see
Ivanov et al., 2001;
Schneider et al., 2003).
N-cadherin, -catenin, and -catenin are expressed in axons
(Murphy-Erdosh, 1994;
Riehl et al., 1996;
Benson and Tanaka, 1998) and at
synapses (Uchida et al., 1996;
Benson and Tanaka, 1998;
Miskevich et al., 1998).
N-cadherin is required for normal axonal targeting and arborization
(Inoue and Sanes, 1997;
Lee et al., 2001), as well as
for normal synapse formation and function
(Tang et al., 1998;
Togashi et al., 2002). We do
not know currently whether -catenin is also required for these
processes. At the synapse, -catenin interacts with at least two
postsynaptic density-95 (PSD-95)/discs large (Dlg)/zona occludens-1 (ZO-1)
(PDZ) proteins, Lin-7/Velis and synaptic scaffolding molecule (S-SCAM)
(Perego et al., 2001;
Nishimura et al., 2002).
Together with cadherins, these proteins recruit and localize proteins at
synaptic junctions (Perego et al.,
2001; Nishimura et al.,
2002). Here we use deletion mutants to examine how disrupting the
interactions mediated by the N- and C-terminal domains of -catenin,
which interact, respectively, with -catenin and synaptic PDZ proteins,
specifically affects stages in axonal arborization.
We have studied -catenin function in axonal arborization of RGCs in
tecta of live Xenopus tadpoles. An advantage of this system is that
the effects of -catenin on arborization of single axons in vivo
can be examined over several days
(O'Rourke and Fraser, 1990;
Cohen-Cory and Fraser, 1995;
Zou and Cline, 1999). We first
show using Xenopus brain sections that -catenin is expressed in
RGC axon arbors. We then express deletion mutants of -catenin in RGCs
and examine their effects on the formation of axonal arbors during development
in vivo. These experiments suggest that interactions mediated by the
N- and C-terminal domains of -catenin, which contain -catenin and
PDZ interaction sites, respectively, initiate and shape the axonal arbors of
RGCs in vivo.
|
Materials and Methods
|
|---|
Xenopus laevis tadpoles. Xenopus laevis embryos were generated by
in vitro fertilization of eggs obtained from females primed with
human chorionic gonadotrophin. Embryos were cultured in a 10% modified
Ringer's solution (MMR) and staged according to Nieuwkoop and Faber
(1956); 0.001%
phenylthiocarbamide was added to the Ringer's solution to reduce pigmentation
(Cohen-Cory and Fraser,
1995).
Antibody staining. Brains with and without green fluorescent
protein (GFP)-expressing retinal ganglion cell (RGC) axon arbors were
dissected from stage 45/46 tadpoles. Tadpoles were anesthetized in a 0.02%
benzocaine solution before dissection. The dissected brains were fixed
overnight at 4°C in a freshly made 4% paraformaldehyde solution and sunk
in sucrose over 2 nights (15 and 30% sucrose in phosphate buffer). The brains
were then embedded in an optimal cutting temperature compound mold, cryostat
sectioned into 30-µm-thick horizontal sections, and mounted onto glass
slides. Antibody staining was performed as follows. Sections were rinsed in
PBS for 5 min, blocked with normal goat serum [5% in PBS plus 0.1% Triton
X-100 (PBST)] for 30 min at room temperature, and incubated overnight at
4°C with a polyclonal rabbit anti--catenin antibody generated
against Xenopus -catenin (1:500 in PBST)
(Kypta et al., 1996). Sections
were then rinsed in PBS three times for 8 min and incubated with a Texas
Red-tagged goat anti-rabbit secondary antibody (1:200 in PBST) (Molecular
Probes, OR) for 1 hr at room temperature. Control sections underwent the same
procedure but were incubated with only the Texas Red secondary antibody.
To determine whether -catenin staining colocalized with GFP axon
arbors, we followed a procedure similar to the one described previously by
Pinches and Cline (1998).
Antibody-stained tectal sections (30 µm) containing a single GFP arbor were
imaged with a confocal microscope (Bio-Rad 600) using a 100x objective
[Zeiss Plan Apochromat; numerical aperture (NA) = 1.40] and a z-series
protocol (z-interval 0.5-1.0 µm). We then opened each optic slice
individually in NIH image (Version 1.62) and traced the portion of the GFP
arbor in that optic slice. We noted all yellow -catenin puncta that
colocalized with the GFP arbor in that particular optic slice. Two criteria
were used to define a yellow spot as a countable puncta. First, in the image
containing the green (GFP)/red (anti--catenin) overlay, we counted only
yellow spots of >1 µm diameter. Second, we counted only those spots with
intensity in the red channel that was twofold greater than that of the average
intensity in the neuropil region of the corresponding control section (stained
only with a secondary antibody). All of the individual tracings for a single
tectal section were combined into one image, and thus the arbor and
corresponding -catenin staining were reconstructed. In all cases, this
constituted a partial reconstruction of the total arbor, comprising only that
portion of arbor present in the 15 µm of the tectal section that had
optimal antibody penetration. In each of these reconstructed GFP arbors, we
counted the total number of branches, measured the length of each of the
branches, and counted the number of -catenin puncta in each of the
branches. From these data, we calculated the average number of -catenin
puncta per 10 µm of branch length. To confirm the specificity of overlap of
red -catenin puncta with the green GFP arbor branches, we mismatched the
red and green images along the y-axis and then calculated the number
of yellow puncta in the mismatched images. On average, mismatched images
contained 33% of the number of yellow puncta as did the correctly matched
images.
Lipofection. To transfect RGCs with DNA constructs, we used the
lipofection protocol first developed for studying axon outgrowth by
Xenopus RGCs (Holt et al.,
1990; Riehl et al.,
1996). Briefly, 24 hr after fertilization, at stages 20-23, we
manually removed vitteline envelopes from embryos and transferred the embryos
to a high salt solution (1x MMR). We back filled micropipettes with a
mixture of DNA and DOTAP lipofection reagent (at a ratio of 1:3) and pressure
injected 50-200 nl of the DNA-DOTAP solution into eye bud primordia. We then
transferred embryos to low-salt 0.1x MMR containing 0.001%
phenylthiocarbamide to inhibit pigmentation. Embryos were raised in the dark
at 23°C for 4 d (until stage 45/46). Expression of proteins has been shown
to peak 4 d after lipofection (Holt et
al., 1990). This corresponds to stage 45/46, by which time RGC
axons have reached the tectum and are forming arbors.
Arbor imaging. At stage 45/46, the heads of tadpoles are
transparent, so that GFP-expressing axon arbors can be imaged in live animals
using fluorescent illumination. For screening and confocal imaging of arbors,
we anesthetized tadpoles in a 0.02% benzocaine solution and then mounted
tadpoles in a chamber made of silicone and sealed with a coverslip. To screen
tadpoles for GFP arbors, we used a 10x objective (Nikon Plan Apo; NA =
0.45) on an upright microscope (Nikon Microphot-FXA). For each tadpole that
contained a GFP arbor we drew a rough sketch of the morphology of the arbor
and its location in the tectum. We confirmed that GFP and
GFPARM-expressing arbors originated from cells located in the
contralateral retina by following several brighter axons back to the
contralateral eye. Animals containing fluorescent arbors were taken to an
inverted confocal microscope (Bio-Rad 600) and imaged using a 40x
objective (Zeiss Plan Neofluor; NA = 0.75). Some of the arbors were confocal
imaged at 24 hr intervals over a 2 or 3 d period. All of the arbors that we
imaged [the thicker, unbranched axons expressing GFPARM and GFP tagged
to the N-terminal domain of -catenin (GFPNTERM), as well as the thinner,
branched arbors expressing GFP, GFPARM and GFP tagged to the C-terminal
domain of -catenin (GFPCTERM)] were located at a relatively superficial
depth within the tectum.
Arbor analysis. Confocal images were projected using the NIH Image
(version 1.62) projection algorithm. From this projected image we counted the
number of branches in the arbors and measured the total length of arbors. By
using the projection algorithm in NIH image, we underestimate both the number
of branch tips and the length of arbors, relative to other studies that
manually reconstructed arbors by tracing each individual slice
(Zou and Cline, 1996).
DNA plasmid construction. The -catenin deletion mutant
ARM contains amino acid residues 1-151 of -catenin fused to
residues 648-781. NTERM contains residues 1-151 of -catenin, and CTERM
contains residues 648-781 of -catenin.
All constructs, including GFP, were subcloned into the Xenopus
expression vector pCS2 [originally constructed by D. Turner (University of
Michigan) and R. Rupp (Max-Planck-Institute, Tuebingen, Germany)]. ARM
and CTERM were constructed previously in our laboratory from a full-length
Xenopus -catenin that had a myc-epitope tag fused to its C
terminus and were subcloned into the expression vector pEGFP-C1 (Clontech)
(Kypta et al., 1996). We
subcloned ARM and CTERM (with GFP at their N termini and myc tags at
their C termini) from pEGFP-C1 into pCS2 using NheI and
XbaI. To make pCS2ARM without GFP we subcloned ARM
(with the myc tag) from peGFP-C1 into pCS2 using EcoRI. To make CTERM
without GFP or a myc tag, we performed PCR on a full-length PBS
Xenopus -catenin (without a myc tag) and subcloned the
resulting PCR fragment into the pCR-Blunt II-TOPO vector. We then subcloned
CTERM (no myc) from pCR Blunt II-TOPO into pCS2 using XbaI.
To make pCS2-GFPNTERM we digested peGFP-C1--catenin (full length)
with XhoI and then subcloned the Xho fragment (containing
the N-terminal domain of -catenin) into peGFP-Cl. We then subcloned
GFPNTERM out of pEGFP-C1 into PCS2 with NheI and XbaI.
|
Results
|
|---|
-catenin is expressed in RGC axon arbors in the tectum
To determine whether -catenin was expressed in RGC axon arbors in
their target tissue, the tectal midbrain, sections from brains of stage 45/46
tadpoles were stained with anti--catenin. Results showed -catenin
expression throughout the neuropil region of the tectum
(Fig. 1A) (tectal
sections from six different brains showed similar staining patterns). Within
the neuropil, -catenin was expressed in a nonuniform pattern, with the
strongest, most dense expression in the lateral neuropil
(Fig. 1A). This
corresponds to the outermost region of the tectum that is first invaded by RGC
axons. In the more medial neuropil, the region of the tectum in which RGC
axons form arbors, the expression of -catenin appeared more sparse and
punctate (Fig. 1A,C).
N-cadherin is also expressed in a similar pattern in the neuropil region of
the Xenopus tectum (Riehl et al.,
1996).
View larger version (87K):
[in this window]
[in a new window]
|
Figure 1. -catenin is expressed in RGC axon arbors in the tectum. Confocal
image (single optic section) of a tectal section taken from a stage 45/46
tadpole stained with anti -catenin antibody (red) shows expression in
the neuropil area, the target region for RGC axons (A). Confocal
image of a GFP-expressing RGC axon arbor (green) in the same optic section
shows that the axon arbor is in the neuropil region of the tectal section
(B). Higher magnification zoom of boxed regions in A and
B shows that -catenin staining is punctate in the neuropil
(C) and that the GFP-expressing RGC axon arbor contains
-catenin puncta (E, arrows, double arrow, arrowhead).
-catenin puncta are located at branch points (D, arrows), in
growth cones (D, double arrow), and along branch shafts (D,
arrowhead). np, Neuropil region of the tectum; cb, cell body region of the
tectum. Scale bars: A, B,10 µm; C-E, 5
µm.
|
|
To examine directly whether RGC axon arbors in the tectum contained
-catenin puncta, single RGC axon arbors were labeled with GFP
(Fig. 1B,D). Tectal
sections containing a single GFP RGC axon arbor were then stained for
-catenin and imaged on a confocal microscope. Examination of these
images revealed specific (yellow) puncta of overlap between the green
GFP-labeled RGC arbors and red-stained -catenin puncta (Figs.
1C-E). The
density of -catenin puncta in RGC axon arbors was 3.4 ± 0.85
-catenin puncta per 10 µm of branch length (440 µm of arbor length
analyzed, 100 µm or more per arbor). Within the RGC axon arbors,
-catenin puncta were found at branch points
(Fig. 1E, arrows),
along shafts of branches (Fig.
1E, arrowhead), and in branch tips
(Fig. 1E, double
arrow). To confirm the specificity of overlap of red -catenin puncta
with the green GFP arbor branches, we mismatched the red and green images
along the y-axis and then compared the number of yellow puncta in the
mismatched images with that in correctly matched red and green images. On
average, mismatched images contained 33% of the number of yellow puncta as
observed in the correctly matched images (see Materials and Methods). In
summary, these data demonstrate that -catenin is expressed in RGC axon
arbors in the tectum. Thus, -catenin may play a role in RGC axon
arborization.
Perturbation of -catenin function does not inhibit RGC axon
outgrowth to the tectum
To perturb -catenin function in RGCs, we used a deletion mutant of
-catenin named ARM that is a fusion of the N-terminal and
C-terminal domains of -catenin (Fig.
2). The N-terminal domain of -catenin contains a highly
conserved subdomain that mediates interactions with -catenin that in
turn associates with the F-actin cytoskeleton
(Ivanov et al., 2001;
Schneider et al., 2003). The
interactions between -catenin and -catenin are required for
strong cadherin-based cell-cell adhesion
(Ivanov et al., 2001). The
C-terminal domain contains a PDZ binding motif that has been shown to interact
with two synaptic PDZ proteins (Perego et
al., 2001; Nishimura et al.,
2002). ARM lacks the central armadillo repeat region of
-catenin that contains interaction sites for cadherins and several
proteins that mediate Wnt signaling, including APC (adenomatous polyposis
coli) and TCF/LEF (T-cell factor/lymphoid enhancing
factor)(Hulsken et al., 1994;
Gottardi and Gumbiner, 2001;
Pokutta and Weis, 2002).
Previous studies have shown that ARM does not activate (or interfere
with) Wnt signaling when expressed in cleavage stage Xenopus embryos
(Funayama et al., 1995;
Sehgal et al., 1997). Because
ARM is expected to compete with endogenous, full-length -catenin
for binding to -catenin and PDZ-containing proteins, we hypothesized
that overexpression of ARM will interfere with recruitment of these
proteins to cadherins, thereby perturbing cadherin-based adhesion and
recruitment of PDZ-containing synaptic proteins (see Discussion).
We first asked whether ARM inhibited RGC axon outgrowth from the
retina to the tectum. To address this issue we examined whether
GFPARM-expressing RGC axons were present in the tecta of stage 45/46
tadpoles. In wild-type animals, a significant number of RGC axons have reached
the tectum by this stage (Holt,
1984). RGCs lipofected with GFPARM extended green
fluorescent axons to the tectum at stage 45/46. To determine whether
GFPARM- and GFP-expressing axons reached the tectum at the same
frequency, we lipofected these two plasmids into two groups of 30 tadpoles. By
stage 45/46, one-third of the tadpoles in each of the two groups showed green
fluorescent RGC axons in the tectum (data not shown). In addition, embryos
injected with ARM in the eyebud of a single, determined hemisphere
extended axons only to the contralateral tectum, similar to embryos expressing
GFP alone (eight embryos analyzed; data not shown). Thus, overexpression of
ARM does not inhibit the extension of RGC axons from the retina to the
tectum. These data are consistent with previous findings showing that
-catenin function is not essential for axon outgrowth
(Loureiro and Peifer, 1998;
Loureiro et al., 2001) and
that overexpression of the region of the cytoplasmic tail of cadherin that
interacts with -catenin is not sufficient to inhibit growth of
Xenopus RGC axons (Riehl et al.,
1996).
catARM expression perturbs axon arborization of RGCs
in vivo in two different ways
Is -catenin function required for axon arborization of RGCs? Before
examining the arbors of RGCs expressing ARM, we imaged and measured
GFP-expressing RGC axon arbors in live tadpoles. At stage 45/46, arbors
containing GFP were moderately branched, with an average branch number of
11 and total arbor length of 300 µm (Figs.
3A,
4A,
5A,B;
Table 1). In a 24 hr period,
these arbors increased their average branch number to 16 and their average
total arbor branch length to 350 µm (Figs.
4A,
5B,C,
Table 1). These axonal
arborization parameters agree with values for wild-type RGC axon arbors at
stage 45/46 reported in a previous study
(O'Rourke and Fraser,
1990).
View larger version (24K):
[in this window]
[in a new window]
|
Figure 3. Axonal arborization phenotypes of RGCs expressing -catenin deletion
mutants. Projected confocal z-series images taken of axon arbors in live
tadpoles show arbor morphologies for RGCs expressing GFP (A) or
GFP-tagged--catenin deletion mutants (B-E). RGC axons that
express the -catenin deletion mutant ARM have two distinct
phenotypes. They either do not arborize (B) or they arborize but
contain longer, tangled branches (C). RGC axons expressing the
-catenin deletion mutant NTERM form severely reduced arbors (D)
that are similar to the subpopulation of ARM arbors with no or few
branches (B). RGC axons expressing CTERM form arbors with long,
tangled branches (E) that are similar to the subpopulation of
ARM arbors with effusive branching (C). RGC axons that express
ARM and NTERM and form severely reduced arbors (B, D) also
have bulbous endings at their axon/branch tips (B, D, arrows) and
appear thicker than RGC arbors that express GFP, ARM, and CTERM
(compare axon/branch thicknesses in B, D with those in A, C,
E). Note that at the level of magnification of these projected confocal
z-series (40x), all of the GFP-tagged--catenin deletion mutant
constructs appear to be uniformly distributed within the axon terminals and
arbors. Scale bars: A-E, 20 µm.
|
|
View larger version (18K):
[in this window]
[in a new window]
|
Figure 5. Quantification of morphological parameters for arbors from RGCs expressing
-catenin deletion mutants confirm phenotypes shown in Figures
3 and
4. The distribution of branch
numbers in arbors at stage 45 shows that GFP axon arbors are distributed about
a mean of 11 branches (A). In contrast, ARM axons are
distributed about two distinct means: ARM arbors either have very few
(1-2) branches or they have approximately the same number of branches as
control GFP arbors (A). In addition, NTERM arbors have no or very few
(1-6) branches, whereas CTERM arbors have numbers of branches comparable with
GFP arbors (A). Plot of mean numbers of branches in arbors over 2 d
shows that GFP arbors acquire more branches over time (B). ARM
arbors with branches and CTERM arbors behave similarly to GFP arbors
(B). In contrast, ARM axons without arbors and NTERM axons
form few or no additional branches over time (B). Branched ARM
arbors (with 9 or more branches) and CTERM arbors also grow faster than GFP
arbors (C) (p < 0.05 for both, Student's t
test). We did not measure the growth rate for unbranched ARM and NTERM
arbors because many RGCs in these two classes did not form arbors
(C). The number of axons analyzed is as follows: A, GFP
(21), ARM (24), NTERM (7), CTERM (10); B, GFP (10),
ARM-branched (6), ARM-unbranched (6), NTERM (7), CTERM (10),
(C) GFP (9), ARM-branched (6), CTERM (10).
|
|
Many axons of RGCs that express ARM do not form arbors
In contrast to RGC axons expressing GFP alone, approximately half of RGC
axons expressing ARM failed to form arbors in the tectum (Figs.
3B,
4B,
5A). At stage 45/46,
these axons had no or very few (at most three) terminal branches (Figs.
3B,
4B,
5A,B). Moreover, when
imaged over several days, we observed that these axons did not form additional
branches with time (Figs.
4B,
5B). The first example
of an axon shown in Figure
4B shows a representative ARM-expressing RGC axon
that initially had two branches; during the following 2 d period, this axon
did not add additional branches. Thus, in a significant fraction of ARM
expressing RGCs, axon branching is severely inhibited.
The axons of RGCs expressing ARM that formed severely reduced arbors
also exhibited other morphological features distinct from those of control
axons of RGCs expressing GFP. First, the axons (and a few secondary branches)
formed by this subpopulation of ARM-expressing RGCs were thicker than
the axons and branches formed by control GFP-expressing RGCs
(Fig. 3, compare A,
B). Second, the tips of the axons and branches formed by
these ARM-expressing RGCs with reduced arborization often terminated in
bulbs or club-like endings (Figs.
3B, arrow,
4B). Both of these
features of ARM-expressing RGC axons became more pronounced over time.
On the second (and third) day of observation, axons of RGCs expressing
ARM became thicker, and the bulbs at the tips of their branches became
larger (Fig. 4B).
Other RGCs overexpressing ARM form misshapen arbors
The remaining ARM-expressing RGC axons that we imaged in tecta of
live tadpoles did form arbors (Figs.
3C,
4C). At stage 45/46,
these arbors had comparable numbers of branches to control arbors of RGCs
expressing GFP alone (Figs.
3C,
4C,
5A,B). The mean number
of branches for ARM arbors with branches was 12, compared with a
mean number of branches of 11 for arbors of controls expressing GFP alone
(Fig. 5A,B). Over a 2
d period, these ARM arbors increased their total branch number, with an
increase comparable with that observed in GFP arbors
(Fig. 5B). Thus in
this group of arbors, branching per se was not inhibited by expression of
ARM.
The arbors of axons expressing ARM, however, formed branches that
were shaped differently than control arbors. First, the total length of axonal
branches of arbors expressing ARM was greater than the length of
control arbors expressing GFP (Figs.
3C,
4C,
Table 1). When examined over
several days, arbors of ARM-expressing RGCs also grew faster than GFP
arbors (Figs. 4C,
5C). Second, the
branches of these arbors were more tangled than branches of control arbors. In
the arbors of RGCs expressing ARM, secondary branches crossed back and
forth several times across the main axis of the arbor
(Fig. 4C, stippling).
In contrast, in control arbors, secondary branches extended straight from the
main axis of arborization and rarely recrossed this axis
(Fig. 4A). Thus, in
this group of RGCs, expression of ARM resulted in accelerated
elongation and more tangled morphology of arbor branches.
The two arborization phenotypes that we observed in
GFPARM-expressing axons were not caused by the inadvertent expression
of GFPARM in neurons that were not RGCs. For both phenotypes we
followed several bright fluorescent axons to their points of origin in the
retinal ganglion cell layer of the contralateral eye. The GFPARM
arborization phenotypes were also not caused by localization of the fusion
protein GFPARM to only a portion of the arbor of RGCs. RGCs
co-lipofected with GFP and ARM in separate plasmids showed the same
arborization phenotypes as did GFPARM-expressing RGCs [n = 10
RGCs co-lipofected; 4 did not arborize, and 6 arborized but had long, tangled
arbors (data not shown)]. Thus, these data implicate -catenin in axon
arborization of RGCs in vivo. They show that -catenin is
required for both the initiation and shaping of RGC arbors.
Overexpression of the N- and C-terminal domain fragments of
-catenin, respectively, inhibit and misshape axon arbors in
vivo
ARM is a fusion of the N-terminal and C-terminal domains of
-catenin. To determine whether the two distinct arborization phenotypes
observed in ARM RGCs reflect distinct activities of these two domains,
we expressed each domain alone in RGCs
(Fig. 2).
RGC axons expressing NTERM had no or severely reduced arborization (Figs.
3D,
4D). At stage 45/46,
NTERM RGC axons had very few branches (one to six branches) (Figs.
3D,
4D,
5A,B). Similar to
ARM axons without branches, NTERM axons did not acquire more branches
over time (Figs. 4D,
5B). Instead, the
average number of branches formed by these neurons was slightly reduced with
time (Fig. 5B).
Moreover, NTERM axons were also thicker than control axons and contained bulbs
at their terminal ends (Figs.
3D, arrow,
4D). Thus, NTERM axons
appeared and behaved very similar to the subpopulation of ARM axons
without arbors (Fig.
3B,D, compare with
Fig. 4B, D).
In contrast, RGCs expressing CTERM formed severely misshapen arbors (Figs.
3E,
4E). At stage 45,
CTERM arbors had numbers of branches comparable with those arbors formed by
RGCs expressing GFP (Fig.
5A,B). Over 2 d, the mean number of branches formed by
RGCs expressing CTERM increased significantly
(Fig. 5B). However,
similar to the arbors of ARM expressing RGCs with extensive branching,
the arbors formed by RGCs expressing CTERM were significantly longer and grew
faster than arbors formed by control RGCs expressing GFP alone (Figs.
3E,
4E,
5C,
Table 1). CTERM arbors were
also very tangled with long branches that crossed and overlapped other
branches of the arbor (Figs.
3E, stippling,
4E). Thus, expression
of CTERM in RGCs generates arbors that resemble those formed by the second
class of RGCs expressing ARM (Figs.
3,
4, compare C, E).
We confirmed that the presence of overextended, misshapen arbors in RGCs
expressing CTERM was not caused by interference of protein interactions by the
GFP and myc tags attached to the CTERM construct. Co-lipofection of a modified
CTERM without the attached GFP and myc tags (see Materials and Methods) and
GFP into RGCs also generated misshapen axon arbors with long, tangled branches
(in three of three RGC axons). Consistent with this finding, recent work in
our laboratory also indicates that attachment of a myc tag at the C-terminal
end of -catenin does not abolish (although it may weaken) PDZ protein
binding to this domain (S. Bamji, N. E. Kimes, and L. F. Reichardt, personal
communication).
These results demonstrate that the N- and C-terminal domains of
-catenin have distinct functions in axon arborization. They suggest that
interactions mediated through the N-terminal domain of -catenin are
required for formation of arbors by RGC axons in the tectum, whereas
interactions mediated by the C-terminal domain are required for normal
extension and shaping of arbors.
RGC axons that express the -catenin deletion mutants
ARM and NTERM are frequently mistargeted within the tectum
Previous studies have shown that in Xenopus tadpoles RGC axons do
not branch until they reach their topographically appropriate locations in the
tectal neuropil (Sakaguchi and Murphey,
1985). This suggests that specific topographic cues are required
to promote formation of axonal arbors by RGCs. Thus, one possibility is that
ARM- and NTERM-expressing RGC axons do not form arbors because they are
mistargeted within the tectum. Examination of the tectal projections of the
subpopulation of ARM-expressing axons without arbors and of
NTERM-expressing axons demonstrated that they did not always terminate in a
topographically appropriate location (Table
2). Two types of targeting errors were observed. The poorly
branched arbors of RGCs expressing ARM or NTERM frequently formed
abnormally short projections into the tectum, or they had unusually long
projections that meandered over the tectum and extended beyond the midline of
the tectum, terminating in the ipsilateral tectum
(Table 2). In contrast, the
well branched arbors formed by RGC neurons expressing GFP, ARM, or
CTERM appeared to project consistently into and terminate within a relatively
central region of the appropriate tectal hemisphere and to thus be correctly
targeted (Table 2). In
conclusion, some of the poorly branched axons of RGCs expressing ARM or
NTERM were clearly mistargeted within the tectum.
|
Discussion
|
|---|
N- and C-terminal domains of -catenin, respectively, initiate
and shape axon arbors in RGCs in vivo
Results presented above show that overexpression of the N- and C-terminal
domains of -catenin, respectively, inhibit and misshape axon arbors in
RGCs in the tecta of live Xenopus laevis tadpoles. These results
suggest that interactions of the N- and C-terminal domains of -catenin
are required, respectively, to form and shape axon arbors. This evidence
implicates two distinct domains of -catenin in two different processes
of axon arborization.
Cadherin-based adhesion and formation of axon arbors in RGCs
Overexpression of -catARM or -catNTERM in RGCs is
expected to interfere with functions mediated by the N-terminal domain of
endogenous -catenin by sequestering proteins that normally bind to this
domain. Results in this paper show that interference with interactions
mediated by this domain prevents formation of axon arbors by RGCs in the optic
tectum. The N-terminal domain of -catenin has been shown to interact
with -catenin, which in turn links the cadherin--catenin complex
to the F-actin cytoskeleton, thereby strengthening cadherin-based cell-cell
adhesion (Ivanov et al., 2001;
Schneider et al., 2003). Thus,
the N-terminal domain of endogenous -catenin may promote axon branching
in RGCs by strengthening cadherin-based cell adhesion. How could strong
cadherin-mediated cell-cell adhesion promote axon arborization in RGCs? We
observed that NTERM- and ARM-expressing RGC axons that did not form
arbors were also frequently mistargeted within the tectum. Other work has
suggested that in Xenopus laevis specific topographic signals in the
tectal target promote branching (Sakaguchi
and Murphey, 1985). Thus, one possibility is that cadherin-based
cell-cell adhesion is required for cell-cell interactions that target RGC
axons to their correct location within the tectum. Consistent with this
possibility, in two different species, cadherin function has been shown to be
required for targeting optic axons within their target tissue
(Inoue and Sanes, 1997;
Lee et al., 2001).
The N-terminal domain of -catenin also contains a glycogen synthase
kinase-3 (GSK-3) binding site, and several phosphorylation sites
that regulate ubiquitination and degradation of the protein
(Schneider et al., 2003). Thus
another possibility for the mechanism of action of the overexpressed
N-terminal domain of -catenin is that it sequesters GSK-3 and the
ubiquitination machinery away from endogenous, full-length -catenin.
This could conceivably reduce the normal level of phosphorylation and
destruction of -catenin, thereby mimicking the action of activated
Wnt-based signaling. However, in a different system, activation of Wnt-based
signaling has been shown to promote, not inhibit, axon branching
(Hall et al., 2000), the
opposite phenotype of what we observe in Xenopus RGCs overexpressing
the N-terminal domain of -catenin.
-catenin-PDZ protein interactions and shaping of RGC axon
arbors
Our results also demonstrate that interactions mediated by the C-terminal
domain of -catenin are required to shape RGC axon arbors. When this
domain was overexpressed in RGCs, longer and more tangled axon branches were
formed by RGCs. These overgrown, tangled axon arbors of RGCs overexpressing
the C-terminal domain of -catenin may correlate with and perhaps be
caused by an inhibition of synaptogenesis. Cadherin function has been shown to
be required for synapse formation in hippocampal neurons
(Togashi et al., 2002).
Introduction of dominant-negative cadherin molecules into cultured hippocampal
cells removed endogenous cadherin and -catenin from synapses, which in
turn dispersed synaptic proteins, disrupted synaptic function, and caused
morphological changes in dendritic spines. Moreover, the last three amino
acids of the C-terminal domain of -catenin comprise a PDZ-binding motif
through which -catenin interacts with, and thereby links cadherin to, at
least two synaptic PDZ proteins (Perego et
al., 2001; Nishimura et al.,
2002). Indeed, overexpression of the C-terminal domain of
-catenin in hippocampal neurons has been shown to inhibit recruitment of
the synaptic PDZ protein S-SCAM (Nishimura
et al., 2002). Consistent with the possibility that the tangled
arbors are caused by an inhibition of synaptogenesis, perturbation of other
proteins involved in synaptogenesis has been shown to also cause excessive
growth of arbors. For example, in the Xenopus retinotectal system,
inhibition of CaMKII (Ca2+/calmodulin-dependent protein
kinase II) function inhibits maturation of synapses and also promotes
excessive growth of axonal arbors (Zou and
Cline, 1999). In addition, in mice lacking either agrin or MuSK
(muscle-specific kinase), the neuromuscular junction fails to differentiate.
In these mice, the axons that innervate the neuromuscular junction also form
extremely abnormal arbors that extend over the entire surface of the muscle
instead of being restricted to a small region, as in normal mice
(Lin et al., 2001).
Several transcriptional coactivators have also been shown to bind to the
C-terminal region of -catenin from armadillo repeat 10 through the
C-terminal end of the protein (Hecht et
al., 1999; Takemaru and Moon,
2000). Thus, another possibility is that the overexpressed
C-terminal domain of -catenin competes with endogenous full-length
-catenin for binding to these transcriptional activators and perturbs
the normal pattern of transcription.
-catARM generates two distinct arborization phenotypes
in RGCs that appear to be regulated by distinct signaling pathways
The deletion mutant -catARM is a fusion of the N- and
C-terminal domains of -catenin. Expression of ARM in RGCs
generated two distinct arborization phenotypes that mimicked the separate
activities of the N- and C-terminal domains of -catenin. These results
suggest that in ARM-expressing RGCs that do not form arbors, binding of
proteins to the N-terminal domain of -catenin is perturbed, whereas in
ARM-expressing RGCs that form extended, misshapen arbors, perturbation
of C-terminal domain interactions is dominant. How does -catARM
result in two very distinctive phenotypes that appear to be caused by
perturbation of two distinct signaling pathways? A likely possibility is that
in the two classes of RGCs, different levels of ARM are present that
selectively perturb distinct protein-protein interactions. In RGCs with lower
levels of ARM, binding of proteins to the C-terminal domain could be
perturbed, resulting in misshapen arbors. In RGCs with higher levels of
ARM, N-terminal (and C-terminal) domain interactions could be
perturbed, thereby inhibiting formation of arbors. Consistent with this
hypothesis, we observed that GFPARM-expressing RGCs that did not form
arbors tended to exhibit brighter GFP fluorescence than
GFPARM-expressing RGCs that formed overextended, misshapen arbors (data
not shown). This suggests that those RGCs that did not form arbors may have
contained higher levels of GFPARM.
Regulation of -catenin-cadherin interactions during axon
outgrowth and arborization in RGCs
Results in this study show that RGC axons expressing -catenin
deletion mutants grow normally from the retina to the tectum
(Loureiro and Peifer, 1998;
Loureiro et al., 2001). In
particular, our data indicate that interactions of the N-terminal domain of
-catenin with proteins such as -catenin are not required for
growth of RGC axons from the retina to the tectum. A previous study showed
that N-cadherin function is required for RGC axon outgrowth to the tectum
(Riehl et al., 1996). However,
this study showed that overexpression of the juxtamembrane domain of
N-cadherin (required for p120 catenin binding) and not the more C-terminal
-catenin binding domain interfered with RGC axon outgrowth
(Riehl et al., 1996).
Consistent with this, our data also indicate that normal RGC axon growth does
not require cadherin interaction with -catenin (and
-catenin).
We also show here that interactions of the N-terminal domain of
-catenin with proteins such as -catenin are required within the
tectum for targeting and arbor formation by RGC axons (see Discussion above).
Cadherin function is also required for targeting and formation of arbors by
RGC axons in the tectum (Inoue and Sanes,
1997). Thus, the cadherin--catenin--catenin adhesion
complex may function together at a later stage to promote targeting and
formation of arbors by RGC axons in the tectum. One mechanism to regulate the
associations and function of the cadherin--catenin--catenin
adhesion complex is through tyrosine phosphorylation of -catenin
(Fujita et al., 2002,
Lilien et al., 2002). Previous
work has suggested that tyrosine phosphorylation of -catenin may mediate
axon outgrowth by RGCs, likely by dissociation of the
cadherin--catenin--catenin adhesion complex
(Kypta et al., 1996;
Lilien et al., 2002). It will
be interesting to test whether dephosphorylation of tyrosine residues on
-catenin is subsequently required during the later developmental events
of targeting and formation of arbors by RGC axons in the tectum.
|
Footnotes
|
|---|
Received Apr. 14, 2003;
revised May. 23, 2003;
accepted May. 23, 2003.
This work was supported by National Research Service Award F32 MH 12613 to
T.M.E. and by the Howard Hughes Medical Institute (HHMI). L.F.R. is an
investigator of the HHMI. We thank members of our laboratory, especially S.
Bamji, L. Elia, and B. Rico, for comments on this manuscript.
Correspondence should be addressed to Tamira Elul, Physiology Department,
University of California San Francisco, 533 Parnassus Avenue, San Francisco,
CA 94143. E-mail:
tamira{at}itsa.ucsf.edu.
Copyright © 2003 Society for Neuroscience
0270-6474/03/236567-09$15.00/0
|
References
|
|---|
Alsins B, Vu T, Cohen-Cory S (2001) Visualizing
synapse formation in arborizing optic axons in vivo: dynamics and
modulation by BDNF. Nat Neurosci 4:
1093-1101.[Web of Science][Medline]
Benson DL, Tanaka H (1998) N-cadherin redistribution
during synaptogenesis in hippocampal neurons. J Neurosci
18: 6892-6904.[Abstract/Free Full Text]
Cantallops I, Haas K, Cline HT (2000) Postsynaptic
CPG15 promotes synaptic maturation and presynaptic axon arbor elaboration in
vivo. Nat Neurosci 3:
1004-1011.[Web of Science][Medline]
Cohen-Cory S, Fraser S (1995) Effects of brain-derived
neurotrophic factor on optic axon branching and remodeling in vivo.
Nature 378:
192-196.[Medline]
Fujita Y, Krause G, Scheffner M, Zechner D, Leddy HE, Behrens J,
Sommer T, Birchmeier W (2002) Hakai, a c-Cbl-like protein,
ubiquitinates and induces endocytosis of the E-cadherin complex. Nat
Cell Biol 4:
222-231.[Web of Science][Medline]
Funayama N, Fagotto F, McCrea P, Gumbiner BM (1995)
Embryonic axis induction by the Armadillo repeat domain of -catenin:
evidence for intracellular signaling. J Cell Biol
128: 959-968.[Abstract/Free Full Text]
Gottardi CJ, Gumbiner BM (2001) Adhesion signaling:
how -catenin interacts with its partners. Curr Biol
11: R792-794.[Web of Science][Medline]
Hall AC, Lucas FR, Salinas PC (2000) Axonal remodeling
and synaptic differentiation in the cerebellum is regulated by WNT-7a
signaling. Cell 100:
525-535.[Web of Science][Medline]
Hecht A, Litterst CM, Huber O, Kemler R (1999)
Functional characterization of multiple transactivating elements of
-catenin, some of which interact with the TATA-binding protein in
vitro. J Biol Chem 274:
18017-18025.[Abstract/Free Full Text]
Holt CE (1984) Does timing of axon outgrowth influence
initial retinotectal topography in Xenopus? J Neurosci
4: 1130-1152.[Abstract]
Holt CE, Garlick N, Cornel E (1990) Lipofection of
cDNAs in the embryonic vertebrate central nervous system.
Neuron 4:
203-214.[Web of Science][Medline]
Hulsken J, Birchmeier W, Behrens J (1994) E-cadherin
and APC compete for the interaction with -catenin and the cytoskeleton.
J Cell Biol 127:
2061-2069.[Abstract/Free Full Text]
Inoue A, Sanes JR (1997) Lamina-specific connectivity
in the brain: regulation by N-cadherin, neurotrophins, and glycoconjugates.
Science 276:
1428-1431.[Abstract/Free Full Text]
Ivanov DE, Philippova MP, Tkachuck VA (2001) Structure
and function of classical cadherins. Biochemistry
66: 1174-1186.[Medline]
Kypta RM, Su H, Reichardt LF (1996) Association
between a transmembrane protein tyrosine phosphatase and the cadherin-catenin
complex. J Cell Biol 134:
1519-1529.[Abstract/Free Full Text]
Lee C, Herman T, Clandinin TR, Lee R, Zipursky SL
(2001) N-cadherin regulates target specificity in the
Drosophila visual system. Neuron
30: 437-450.[Web of Science][Medline]
Lilien J, Balsamo J, Arregui C, Xu G (2002) Turn-off,
drop-out: functional state switching of cadherins. Dev Dyn
224: 18-29.[Web of Science][Medline]
Lin W, Burgess RW, Dominguez B, Pfaff SL, Sanes JR, Lee KF
(2001) Distinct roles of nerve and muscle in postsynaptic
differentiation of the neuromuscular synapse. Nature
410: 1057-1064.[Medline]
Loureiro J, Peifer M (1998) Roles of Armadillo, a
Drosophila catenin, during central nervous system development.
Curr Biol 8:
622-632.[Web of Science][Medline]
Loureiro JJ, Akong K, Cayirlioglu P, Baltus AE, DiAntonio A, Peifer
M (2001) Activated armadillo/-catenin does not play a
general role in cell migration and process extension in Drosophila.
Dev Biol 235:
33-44.[Medline]
Miskevich F, Zhu Y, Ranscht B, Sanes JR (1998)
Expression of multiple cadherins and catenins in the chick optic tectum.
Mol Cell Neurosci 12:
240-255.[Medline]
Murphy-Erdosh C (1994) The molecular function and
embryonic expression of B-cadherin and cadherin associated proteins.
PhD thesis, University of California San Francisco.
Nieuwkoop PD, Faber J (1956) Normal table of
Xenopus laevis. Amsterdam: Daudin.
Nishimura W, Yao I, Iida J, Tanak N, Hata Y (2002)
Interaction of synaptic-scaffolding molecule and -catenin. J
Neurosci 22:
757-765.[Abstract/Free Full Text]
O'Rourke NA, Fraser SE (1990) Dynamic changes in optic
fibres terminal arbors lead to retinotectal map formation: an in vivo
confocal microscopic study. Neuron 5:
159-171.[Web of Science][Medline]
Perego C, Vanoni C, Massari S, Longhi R, Pietrini G
(2001) Mammalian Lin-7 PDZ proteins associate with -catenin
at cell-cell junctions of epithelia and neurons. EMBO J
19: 3978-3989.[Web of Science]
Pinches E, Cline HT (1998) Distribution of synaptic
vesicle proteins within single retinotectal axons of Xenopus
tadpoles. J Neurobiol
426-434.
Pokutta S, Weis WI (2002) The cytoplasmic face of cell
contact sites. Curr Opin Struct Biol 12:
255-262.[Web of Science][Medline]
Riehl R, Johnson K, Bradley R, Grunwald GB, Cornel E, Lillienbaum
A, Holt CE (1996) Cadherin function is required for axon
outgrowth in retinal ganglion cells in vivo. Neuron
17: 837-848.[Web of Science][Medline]
Sakaguchi DS, Murphey RK (1985) Map formation in the
developing Xenopus retinotectal system: an examination of ganglion
cell terminal arborizations. J Neurosci
5: 3228-3245.[Abstract]
Schneider SQ, Finnerty JR, Martindale MQ (2003)
Protein evolution: structure-function relationships of the oncogene
-catenin in the evolution of multicellular animals. J Exp
Zool 295B:
25-44.
Sehgal RN, Gumbiner BM, Reichardt LF (1997) Antagonism
of cell adhesion by an -catenin mutant, and of the Wnt-signaling
pathway by -catenin in Xenopus embryos. J Cell
Biol 139:
1033-1046.[Abstract/Free Full Text]
Takemaru KI, Moon RT (2000) The transcriptional
coactivator CBP interacts with -catenin to activate gene expression.
J Cell Biol 149:
249-254.[Abstract/Free Full Text]
Tang L, Huang CP, Schuman EM (1998) A role for the
cadherin family of cell adhesion molecules in hippocampal long-term
potentiation. Neuron 20:
1165-1175.[Web of Science][Medline]
Togashi H, Abe K, Mizoguchi A, Takaoka K, Chisaka O, Takeichi M
(2002) Cadherin regulates dendritic spine morphogenesis.
Neuron 35:
77-89.[Web of Science][Medline]
Uchida N, Honjo Y, Johnson KR, Wheelock MJ, Takeichi M
(1996) The cadherin/catenin adhesion system is localized in
synaptic junctions bordering transmitter release zones. J Cell
Biol 135:
767-779.[Abstract/Free Full Text]
Wang KH, Brose K, Arnott D, Kidd T, Goodman CS, Henzel W,
Tessier-Lavigne M (1999) Biochemical purification of a mammalian
slit protein as a positive regulator of sensory axon elongation and branching.
Cell 96:
771-784.[Web of Science][Medline]
Yates PA, Roskies AL, McLaughlin T, O'Leary DDM (2001)
Topographic-specific axon branching controlled by Ephrin-A is the critical
event in retinotectal map development. J Neurosci
21: 8548-8563.[Abstract/Free Full Text]
Zou DJ, Cline HT (1996) Expression of constitutively
active CaMKII in target tissue modifies presynaptic axon arbor growth.
Neuron 16:
529-539.[Web of Science][Medline]
Zou DJ, Cline HT (1999) Postsynaptic
calcium/calmodulin-dependent protein kinase II is required to limit
elaboration of presynaptic and postsynaptic neuronal arbors. J
Neurosci 19:
8909-8918.[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
M. D. David, A. Yeramian, M. Dunach, M. Llovera, C. Canti, A. G. de Herreros, J. X. Comella, and J. Herreros
Signalling by neurotrophins and hepatocyte growth factor regulates axon morphogenesis by differential {beta}-catenin phosphorylation
J. Cell Sci.,
August 15, 2008;
121(16):
2718 - 2730.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Bahmanyar, D. D. Kaplan, J. G. DeLuca, T. H. Giddings Jr., E. T. O'Toole, M. Winey, E. D. Salmon, P. J. Casey, W. J. Nelson, and A. I.M. Barth
{beta}-Catenin is a Nek2 substrate involved in centrosome separation
Genes & Dev.,
January 1, 2008;
22(1):
91 - 105.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. P. Meyer and S. J Smith
Evidence from in vivo imaging that synaptogenesis guides the growth and branching of axonal arbors by two distinct mechanisms.
J. Neurosci.,
March 29, 2006;
26(13):
3604 - 3614.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Votin, W. J. Nelson, and A. I. M. Barth
Neurite outgrowth involves adenomatous polyposis coli protein and {beta}-catenin
J. Cell Sci.,
December 15, 2005;
118(24):
5699 - 5708.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Xiao, T. Roeser, W. Staub, and H. Baier
A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection
Development,
July 1, 2005;
132(13):
2955 - 2967.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
