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Fig. 1 Expression of netrin-1 and of its receptors DCC and UNC-5 in stage 45 Xenopus optic
tectum. a Schematic of coronal section of Xenopus retinotectal circuit. RGC axons (green)
travel from the contralateral eye to connect with tectal neurons in the neuropil (blue). b, c In
situ hybridization with Xenopus-specific antisense netrin-1 probes. Coronal sections of the
midbrain at the level of the optic tectum show ventral-high (double arrows) to dorsal-low
(arrow) netrin-1 mRNA expression along the ventricle wall. dâg Coronal and h, i horizontal
sections show DCC and UNC-5 expression. dâg Co-immunostaining illustrates the
differential distribution of UNC-5 (red) and DCC (green) immunoreactivity. d DCC
immunoreactivity (green) is localized to the cell bodies in the dorsal tectum and proximal
dendrites and to incoming axons near the dorsal neuropil (arrow). The tectal neuropil (np) is
also positive for DCC. The low- (e, f) and high- (g) magnification coronal images show
UNC-5 (red) and DCC (green) co-localization, with UNC-5 being localized to a subset of
cells that also expresses DCC (g, arrowheads). f Counterstaining with DAPI (blue) serves to
distinguish nuclear staining from cytoplasmic UNC-5 (red) and DCC (green) expression in
tectal cells. h UNC-5 immunoreactivity (green) is localized to a subset of cell bodies in the
dorsal area of the tectum and area adjacent to the tectal neuropil identified by
immunostaining with antibodies to the presynaptic protein SNAP-25 (red). i Anterograde
labeling with rhodamine dextran shows that RGC axons (red) terminate in the areas of the
tectal neuropil (arrow) where UNC-5 immunopositive neurons localize (green). D dorsal, V
ventral, C caudal, R rostral, L lateral, np neuropil. Scale bars: 50 μm in bâf, 20 μm in g, 20
μm in hâi
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Fig. 2 Specific patterns of DCC and UNC-5 expression in the X. laevis central nervous
system. Immunostaining with antibodies to UNC-5 (red) and DCC (green) revealed specific
patterns of expression of the netrin-1 receptors in stage 45 tadpoles. aâg UNC-5 (red) and
DCC (green) immunoreactivity in the forebrain (a), pre-tectum (b), caudal tectum (e),
hindbrain (f), and rostral spinal cord (g) demonstrate a specific pattern of expression for each
of these receptors within subpopulations of neurons in the central nervous system. c UNC-5
immunostaining (red) localizes to subpopulations of neurons in the dorsal tectum, lateralventral
midbrain, ventral midline (vm), and infundibulum (if). d DCC immunoreactivity
(green) is localized in dorsal tectal neuron cell bodies and processes in the tectum and ventral
midline, as well as in the tectal neuropil (np). e, f Note the specificity of immunostaining and
co-localization of UNC-5 and DCC expression in subpopulations of cells in the caudal tectum
(e) and hindbrain (f) and the localization of DCC receptors to discrete fiber tracts (arrows). g,
h UNC-5 (red) and DCC (green) immunoreactivity in the rostral (g) and caudal (h) spinal
cord is localized to fiber tracts and ventral midline in agreement with published observations
in Xenopus and other species (for review, see [42, 5, 43â45]). DCC immunoreactivity in the
spinal cord is similar when staining with antibodies directed against the extracellular (g) or
intracellular (h, bottom) domains of DCC. Counterstaining with DAPI (blue) serves to
distinguish nuclear staining from UNC-5 (red) and DCC (green) expression in cell bodies
and fiber tracts. Scale bars: 50 μm
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Fig. 3 Protein diffusion after treatment. a Schematic of coronal view of stage 45 Xenopus
retinotectal circuit depicting injection sites (red arrows) and spread of injected proteins
(violet color). b Coronal section at the level of the optic tectum immunostained with
antibodies to netrin-1. Note endogenous netrin immunoreactivity in cell body layer and
neuropil. c–g Sections at the level of the optic tectum of tadpoles injected with vehicle,
recombinant netrin-1, UNC5H2-Ig, or anti-DCC were immunostained to examine the spread
of the injected proteins after treatment. c Quantitative analysis of fluorescence intensity in
sections of uninjected tadpoles (Endogenous Netrin) or tadpoles injected with recombinant
netrin (Injected rNetrin-1). The relative levels of netrin within the cell body layer and the
neuropil are illustrated by the average pixel intensity values along the medial-to-lateral axis
of the tectum. The zero value in the X-axis corresponds to the cell body layer-neuropil
boundary; negative X-coordinates represent distance from the boundary to the ventricle while
positive X-coordinates represent distance from the boundary to the lateral-most neuropil. n =
10 brain sections per group, from four tadpole brains per group, with three 20-pixel-wide line
scans quantified per section. Error bars represent the standard error of the mean. d–g Sample
coronal sections of tadpoles injected with vehicle (d), recombinant netrin-1 (e), UNC5H2-Ig
(f), or anti-DCC (g) immunostained with chick antibodies to netrin-1 and Alexa 488
secondary antibodies to chick IgG (top; d, e) or stained with Alexa 488 secondary antibodies
to human IgG (top; f) or mouse IgG (top; g). The pseudo-color images in d–g (bottom) show
the relative intensity of the Alexa fluor 488 fluorescence. Pixel intensity values ranged from 0
(black) to 255 (white) as illustrated by the color-scale bar (d, bottom). Note the increased
immunofluorescence in the cell body layer and neuropil of netrin-1-treated tadpoles (e) when
compared to vehicle-injected controls (d) and with endogenous netrin-1 expression (b). In f
and g, the relatively higher fluorescence intensity in the hemisphere that received the
injection (red arrows) and the diffusion patterns of the proteins are more evident in the
pseudo-color images. In g, white arrows point to fluorescently labeled cells in the injected
tectal hemisphere. Scale bars in b, d–g: 50 μm
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Fig. 4 Rapid remodeling of dendritic arbors upon acute manipulations in netrin signaling. a
Schematic diagram of a stage 45 Xenopus tectal midbrain (horizontal view). Tectal neurons
(red) make dendritic connections with contralateral RGC axons (green) within the tectal
neuropil. b, c Sample RGC axons and tectal neurons, visualized by expression of GFP and
tdTomato, respectively, in control (b) and netrin-treated (c) tadpoles. Note change in tectal
neuron dendritic architecture evident at 4 and 24 h after netrin-1 treatment (inserts). dâg
Confocal projections of representative tectal neurons co-expressing tdTomato (red) and
PSD95-GFP (green) in tadpoles injected with control vehicle solution (d), Netrin (e),
UNC5H2-Ig (f), or Netrin + UNC5H2-Ig (g). Note the emergence of an alternative primary
dendrite (arrow) growing towards the midline in neurons exposed to netrin-1 or UNC5H2-Ig.
Tadpoles treated with netrin + UNC5H2-Ig appeared identical to controls. Axons of tectal
neurons are labeled by the asterisks. Scale bars: 20 μm
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Fig. 5 Altering endogenous netrin levels decreases dendrite branch number and total dendritic
arbor length. Effects of tectal microinjection of netrin, UNC5H2-Ig, or netrin + UNC5H2-Ig
on total dendrite branch number (a) and length (b). Netrin-1 and UNC5H2-Ig altered tectal
neuron morphology with a different time scale. Note that exogenous netrin-1 treatment
decreased dendrite arbor length at 24 h, while the UNC5H2-Ig treatment that sequesters
endogenous netrin induced a transient but significant decrease in branch number at the 0- to
2- and 0- to 4-h imaging intervals when compared to all other treatments. Co-treatment with
netrin + UNC5H2-Ig did not influence branch number or length. Values are expressed as
percent change from the initial 0-h imaging session. Two-way ANOVA with Bonferroni
multiple comparison test; *p < 0.05, **p < 0.01. Error bars indicate SEM
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Fig. 6 Blocking DCC signaling induces changes in dendritic arbor shape without altering
total branch number or length. aâc Confocal projections of representative tectal neurons coexpressing
tdTomato (red) and PSD95-GFP (green) in tadpoles injected with control vehicle
solution (a), netrin-1 (b), or function-blocking antibodies to DCC (c). While control neurons
branch, elaborate, and add PSD95-GFP puncta (a), neurons in tadpoles treated with netrin-1
undergo dynamic remodeling of existing branches (b). Short arrows point to dendrites with
altered directions of growth. Neurons in tadpoles treated with anti-DCC (c) also appear to
change dendritic arbor direction and form small basal projections at 2 and 4 h post-injection
(long arrows). Scale bars: 20 μm. d, e Comparison of effects of netrin and anti-DCC on total
branch number (d) and dendritic arbor length (e). Note that only netrin-1 treatment decreased
arbor length at 24 h (e), but neither netrin nor anti-DCC affects the total number of branches
(d). Two-way ANOVA with Bonferroni multiple comparison test; *p < 0.05. Error bars
indicate SEM
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Fig. 8 Altered netrin-1 levels and DCC signaling impact postsynaptic cluster remodeling. aâd
Confocal projections of single branches from representative tectal neurons co-expressing
tdTomato (red) and PSD95-GFP (green) from control (a), netrin (b), UNC5H2-Ig (c), or
Anti-DCC (d) groups before and after treatment. Dynamic remodeling of postsynaptic
specializations is illustrated by the addition (green arrowheads) and elimination (yellow
arrowheads) of PSD95-GFP clusters. Blue arrowheads denote puncta that remained stable
from one observation interval to the next; white arrowheads denote puncta that were present
at the initial observation time point but were eliminated (yellow) at 2 h. Scale bar: 20 μm
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Fig. 9 Postsynaptic cluster addition and stabilization are modulated by alterations in netrin
signaling. a, b Effects of netrin-1, UNC5H2-Ig, or anti-DCC treatments on postsynaptic
cluster remodeling were quantified as the proportion of PSD95-GFP puncta that were added
(a) and remained stable (b) within the 0â2 and 2â4 observation intervals. Note that
significantly more PSD95-GFP puncta were between 0 and 2 h (a), while fewer were stable
between 2 and 4 h (b) following netrin-1 or anti-DCC treatment when compared to controls. c
To determine the relative stability of newly added postsynaptic clusters, we quantified
relative proportion of PSD95-GFP puncta added over the 0- to 2-h interval that were lost in
the subsequent 2- to 4-h interval for a subset of randomly selected neurons for each group (n
= 4). PSD95-GFP puncta added from 0 to 2 h were significantly less stable in the netrin-1- or
anti-DCC-treated neurons. Statistical significance was by one-way ANOVA and with
unpaired t-tests. Significance when compared to control is *p < 0.05, **p < 0.01. Error bars
indicate SEM
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Fig. 10 Overlays of sample neurons at 0, 2, and 4 h illustrate changes in dendritic arbor
morphology in response to treatment and between imaging intervals. a Confocal stacks of
individual neurons from control, netrin-1-, UNC5H2-Ig-, and anti-DCC-treated tadpoles were
reconstructed with MetaMorph creating three-dimensional wireframes of each stack.
Wireframes were color-coded based on imaging time point (black, 0 h; blue, 2 h; red, 4 h),
overlapped, and aligned over Scholl concentric circles with the primary dendrite placed at a
0° angle (X-axis; gray line). Dynamic changes in dendritic morphology every 2 h over a 4-h
imaging period are illustrated by the emergence of blue (2 h) or red branches (4 h) from
under the black wireframe (0 h). b, c Cumulative wireframes from a subset of seven neurons
per condition better illustrate the dynamic changes in growth between the 0- and 2-h imaging
interval (b), and the 0- and 4-h imaging interval (c), for each treatment group. Large arrows
point to sample ectopic branches newly extended at the time point indicated by the color of
the arrow (blue, 2 h; red, 4 h). Short arrows point to already established branches that
changed their directionality of growth at the time point indicated by the color of the arrow
(blue, 2 h; red, 4 h)
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Fig. 11 Perturbations in tectal netrin levels or signaling alter dendritic arbor directionality. a
Proportion of neurons that developed ectopic basal projections within the 24-h period in each
group. b Angle analysis performed on tectal neuron arbors sums all branch points to produce
a net vector. The angle change was calculated from the tangents of arbors from 0 to 4 h. c
The change in dendritic arbor directionality is shown as the difference in angle for neurons
from 0 to 4 h and was measured both including (with) and excluding (without) ectopic
projections. d Proportion of stable branches with net angle change. The percentage of stable
branches that individually changed their angle by at least 10° was calculated for a subset of
randomly selected neurons (n = 4). The individual branch tip vectors for each branch were
compared from 0 to 4 h to calculate the angle change. Note that a larger proportion of stable
branches altered their angle in neurons following anti-DCC treatment when compared to all
other groups. Statistical significance was by Kruskal-Wallis Friedman with Dunnâs multiple
comparison test. Significance when compared to control is *p < 0.05, **p < 0.01, ***p <
0.001. Error bars indicate SEM
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Fig. 12 Individual branches change their orientation of growth in response to altered netrin
levels. a, b The maximum projections of each confocal z-stack of two sample neurons at the
0-, 2-, and 4-h imaging time points, and the corresponding 90° view of each threedimensional
z-stack, illustrate the dynamic changes in growth and directionality of individual
dendrites in response to acute netrin-1 treatment. The neuron in a corresponds to that shown
in Fig. 5b. bâ For the sample neuron in b, a single primary dendrite and its individual
secondary branches of the same neuron can be discerned in the higher magnification images
by selecting and projecting only the z-planes from each confocal stack that include that
branch. By isolating the individual dendrite from the rest of the dendritic arbor, one can better
differentiate the change in the direction of growth of the primary dendrite (short white
arrows) that took place while some of its secondary branches were pruned (double blue
arrows) or changed their direction of growth (green arrow) and others were maintained.
Scale bars: 20 μm
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Fig. 13 Sequestration of endogenous netrin-1 with UNC-5 ectodomain affects swimming
behavior in a visually guided task. a Schematic of the visual avoidance task viewed from
above. Stage 45 tadpoles swim in the 60-mm open field (blue arrow and dotted line) while
the Matlab program projects an image on the monitor where the petri dish rests. The black
line outside the field represents the vector the 0.3-mm dot (small black circle) will travel.
Every 30 s, the 0.3-mm dot appears in the center and is directed towards the black line to
intercept the tadpole (black arrow). The tadpole’s response to the advancing stimuli (gray
circle) is video recorded and typically results in the tadpole changing its swimming velocity
and/or direction (red arrows). b Reaction to the presentation of a moving visual stimulus for
tadpoles before treatment (0 h) and 4 h after treatment with vehicle solution (control), netrin-
1, anti-DCC, or UNC5H2-Ig is shown as the percent of trials in which tadpoles showed an
avoidance response. Tadpoles injected with UNC5H2-Ig had decreased avoidance responses
to the presentation of the stimulus 4 h post-injection. Two-way, repeated measures ANOVA
with Bonferroni multiple comparison test; *p < 0.05, **p < 0.01. Error bars indicate SEM
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Fig. 7 Acute manipulations in endogenous netrin levels induce rapid changes in dendrite
remodeling. a, b Effects of netrin-1, UNC5H2-Ig, or anti-DCC treatments on new branch
addition (a) and branch stabilization (b). Note that while netrin-1 and UNC5H2-Ig increased
branch addition and decreased branch stabilization throughout the 24-h imaging period, the
anti-DCC treatment influenced the stability of branches at the 4- to 24-h interval only. c
Relative proportion of neurons with different branch addition rates. A significant shift in the
distribution of neurons that responded with increased branch addition rates was observed
after netrin-1 and UNC5H2-Ig treatments. Values are expressed as percent change from total
branches. d Relative change in DCI values is shown for each group at all imaging intervals.
Note that neurons in UNC5H2-Ig-treated tadpoles significantly decreased their complexity by
4 h compared to controls. Two-way ANOVA with Bonferroni multiple comparison test; *p <
0.05, **p < 0.01, ***p < 0.001. Error bars indicate SEM
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