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The Journal of Neuroscience, November 1, 2001, 21(21):8548-8563
Topographic-Specific Axon Branching Controlled by Ephrin-As Is
the Critical Event in Retinotectal Map Development
Paul A.
Yates,
Adina L.
Roskies,
Todd
McLaughlin, and
Dennis D. M.
O'Leary
Molecular Neurobiology Laboratory, The Salk Institute, La Jolla,
California 92037
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ABSTRACT |
The retinotectal projection is the predominant model for studying
molecular mechanisms controlling development of topographic axonal
connections. Our analyses of topographic mapping of retinal ganglion
cell (RGC) axons in chick optic tectum indicate that a primary role for
guidance molecules is to regulate topographic branching along RGC
axons, a process that imposes unique requirements on the molecular
control of map development. We show that topographically appropriate
connections are established exclusively by branches that form along the
axon shaft. Initially, RGC axons overshoot their appropriate
termination zone (TZ) along the anterior-posterior (A-P) tectal axis;
temporal axons overshoot the greatest distance and nasal axons the
least, which correlates with the nonlinear increasing A-P gradient of
ephrin-A repellents. In contrast, branches form along the shaft of RGC
axons with substantial A-P topographic specificity. Topography is
enhanced through the preferential arborization of appropriately
positioned branches and elimination of ectopic branches. Using a
membrane stripe assay and time-lapse microscopy, we show that branches
form de novo along retinal axons. Temporal axons
preferentially branch on their topographically appropriate anterior
tectal membranes. After the addition of soluble EphA3-Fc, which blocks
ephrin-A function, temporal axons branch equally on anterior and
posterior tectal membranes, indicating that the level of ephrin-As in
posterior tectum is sufficient to inhibit temporal axon branching and
generate branching specificity in vitro. Our findings
indicate that topographic branch formation and arborization along RGC
axons are critical events in retinotectal mapping. Ephrin-As inhibit
branching along RGC axons posterior to their correct TZ, but alone
cannot account for topographic branching and must cooperate with other
molecular activities to generate appropriate mapping along the A-P
tectal axis.
Key words:
axon guidance; axon repellents; branch inhibition; chick; Eph receptors; EphA3-Fc; gradients; membrane stripe assay; time-lapse
imaging; topographic maps
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INTRODUCTION |
The projection of retinal ganglion
cell (RGC) axons to the optic tectum, or its mammalian homolog, the
superior colliculus (SC), has been a model for studying the development
of topographic connections. EphA receptors and their ephrin-A ligands
are the only molecules described that meet criteria for topographic
guidance molecules (Flanagan and Vanderhaeghen, 1998 ; O'Leary et al.,
1999) established by Sperry (1963) in the chemoaffinity hypothesis. In
chick, ephrin-A2 and ephrin-A5 combine to form an increasing anterior
(A) to posterior (P) gradient across tectum, and their receptor, EphA3,
is expressed by RGCs in an increasing nasal to temporal gradient (Cheng
and Flanagan, 1994; Cheng et al., 1995; Drescher et al., 1995; Monschau
et al., 1997; Connor et al., 1998). These expression patterns correlate
with the mapping of the temporal-nasal retinal axis along the A-P
tectal axis and the demonstrations that ephrin-As preferentially repel
temporal axons (Nakamoto et al., 1996; Monschau et al., 1997; Frisen et
al., 1998). Genetic analyses in mice show that ephrin-A2 and ephrin-A5
are required for the proper mapping of RGC axons in the SC (Frisen et
al., 1998; Feldheim et al., 2000) and that EphA receptors mediate their repellent action (Brown et al., 2000).
Defining how RGCs develop topographic connections is critical for
defining the roles of guidance molecules, creating accurate models of
this process, and determining whether additional activities are
required. Surprisingly, however, the development of topographic projections in the chick tectum and mammalian SC remains poorly defined
and controversial. Some studies have concluded that the topographic
targeting of RGC growth cones is the primary mechanism for map
development in chick tectum and cat SC (Thanos and Bonhoeffer, 1987;
Chalupa et al., 1996; Chalupa and Snider, 1998). Other studies report
that the initial projection to the chick tectum or rat SC is
topographically diffuse and that many temporal axons make targeting
errors and form branches and arbors at topographically incorrect sites
(O'Leary et al., 1986; Nakamura and O'Leary, 1989; Simon and
O'Leary, 1992a,b).
The goal of the present study was to define mechanisms that RGCs use to
develop their topographic projection to the tectum in chicks by
quantifying topographic specificity in growth cone targeting, axon
branching, and arborization. We chose the chick retinotectal projection
because it has been the preeminent system for the molecular analysis of
RGC axon mapping. We show that essentially all RGCs initially overshoot
the location of their future termination zone (TZ) along the A-P tectal
axis and establish topographic connections by the arborization of
branches that form along the axon shaft. Although RGC growth cones fail
to target their appropriate TZ, axon branching exhibits a high degree
of topographic specificity along the A-P tectal axis. In
vitro, we show that temporal axons exhibit topographic branching
and that the level of ephrin-A ligands in posterior tectum is
sufficient to inhibit branching along temporal axons. Time-lapse
microscopy was used to investigate mechanisms of branching specificity
in vitro. Because topographic specificity in growth cone
targeting and axon branching pose different requirements on their
molecular control, our findings have substantial implications for the
roles and limitations of ephrin-As in map development. In addition,
they provide a framework for modeling the molecular control of RGC axon mapping.
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MATERIALS AND METHODS |
Animals
Embryos of a White Leghorn strain of chickens were raised from
fertile eggs in a high-humidity forced-draft incubator at 38°C. Eggs
were windowed on embryonic day (E) 3 of incubation, and the hole was
sealed with transparent tape until tracer application. Embryos were
staged according to the criteria of Hamburger and Hamilton (1951) at
the time of tracer injection and fixation, as well as when tissue was
collected for in vitro assays.
In vivo analysis
RGC axons were labeled by a discrete pressure injection of
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes, Eugene, OR), or in a few cases with a crystal
placement of 3,3'-dioctadecyloxycarbocyanine perchlorate (DiO;
Molecular Probes), into the retina on E9-E12 using a Picospritzer (General Valve, Fairfield, NJ) and returned to the incubator. Embryos
were perfused transcardially with 4% paraformaldehyde 12-72 hr later.
The retina and contralateral tectum were whole mounted and scanned
using a custom macro on a Bio-Rad 1024 confocal microscope attached to
a Zeiss inverted microscope using a 20 or 25× lens for the tectum and
a 6.3 or 10× lens for the retina. Confocal images were projected in
three dimensions (3D) and montaged using custom macros written for NIH
Image. In some cases, the whole mounts were analyzed and photographed
on an upright fluorescence microscope using RITC (DiI) or FITC (DiO)
filter cubes.
In our initial analyses of the patterns of branching and growth cone
targeting, injections of DiI were made in peripheral temporal retina at
locations 5-10% of the total distance along the temporal-nasal axis.
Quantitative analyses were performed only on localized injections
labeling 5-15 axons with branches that could be unambiguously
identified. Confocal microscopy and subsequent projections in 3D
allowed the axonal origin of branches to be resolved definitively. In a
small number of older cases the entire extent of arborization in a
dense TZ could not be resolved completely. Axons, branches, and arbors
were digitally traced on the scanned confocal montages, and the traced
image was analyzed using another custom NIH Image macro. This macro
determined the lengths, locations, and connections of all axons,
branches, and arbors within the tectum. Interstitial arbors were
defined as interstitial branches originating from the axon shaft that
had one or more secondary branches. Terminal arbors were defined by a
similar criteria, but their origin was located within 250 µm of the
distal end of the primary axon shaft. By E12 and E13, elimination of
the overshooting segment of the primary axon positioned the distal end
of some axons close enough to the TZ that some proportion of arbors
that had initially developed as interstitial arbors were incorrectly
scored as terminal arbors.
Additional quantitative analyses were performed using a custom macro
written in Microsoft Excel Visual Basic with the data provided by the
NIH Image macro. Distribution of axons, branches, and arbors was
analyzed using composite projections from 7-10 tecta for each age.
Analyses of these distributions were made with respect to the
topographically correct TZ in the tectum, which was determined,
independent of the distribution of labeled axons in the tectum, by
mapping the injection site in the retina onto the tectum. We measured
the distance of the DiI injection site from the temporal edge of the
retina and expressed this value as a percentage of the total distance
across the temporal-nasal axis. The predicted TZ was then placed at
the same percentage distance from the anterior edge of the tectum
relative to the total distance across the A-P axis of the tectum. In
E13 embryos, the predicted TZ was always located at the A-P position of
the emerging TZ, thus confirming the accuracy of this mapping
procedure. The TZ was defined as a zone extending 250 µm both
anterior and posterior to this predicted point in tectum and was chosen
because this corresponded to the typical 500 µm width of mature
retinal arborizations in tectum (Thanos and Bonhoeffer, 1987; Nakamura and O'Leary, 1989).
Average branch density outside the TZ was determined by dividing the
total number of branches outside the TZ at each age by the average
length of labeled axons multiplied by the total number of labeled axons
at each age. We subtract 500 µm from the length of axons that project
past the TZ when determining average axon length used in the
calculation above so that segments of the axon located in the TZ were
not included. Average branch density posterior to the TZ was determined
for each age by dividing the total number of branches posterior to the
TZ by the average overshoot for axons that project past the TZ
multiplied by the number of axons projecting past the TZ.
Analysis of the differential, position-dependent overshoot of the TZ
was performed by making DiI injections at a number of locations in
temporal, central (dorsal), and nasal retina at E9, E10, E11, and E12;
labeled RGC axons were analyzed in tectal whole mounts 1 d later.
Average overshoot was quantified using E11 tectal whole mounts at
similar developmental states, assessed by the degree of branching and
arborization. Overshoot for peripheral temporal axons was measured at
early E11, whereas overshoot for peripheral nasal axons was measured at
late E11 because there is a developmental delay for RGC axons
originating from more peripheral nasal locations compared with more
central or temporal locations in the retina. To verify that the maximum
overshoot was measured, the overshoot was also determined at E10, E12,
and E13. Nasal axons had not yet reached their TZ at E10, although at
E12 and E13 we found less overshoot for all retinal locations as
compared with E11 (data not shown). Average overshoot shown in Figure 5 was quantified only for RGC axons that had either reached or projected past the predicted TZ. Growth cones anterior to the predicted TZ were
not included because this would bias the analysis, grossly underestimating the overshoot for more nasal axons, because many nasal
axons are still extending across the tectum at this age. Statistical
analysis of branch, axon, and arbor distributions was performed using
Statview. Quantitative analysis was performed on the following data:
number of tectal whole mounts: E10 (10), E11(7), E12 (9), E13 (7);
number of axons: E10 (104), E11 (64), E12 (68), E13 (52); number of
branches: E10 (302), E11(248), E12 (241), E13 (141).
In vitro membrane branching assays
Assay preparation. The membrane stripe assay (Walter
et al., 1987a,b) was used in a modified form (Roskies and O'Leary,
1994). Tecta from E9-E10 chick embryos were used for the preparation of membrane carpets. The brain was dissected from the skull, the pia
was removed, and the tecta were dissected into thirds. The middle
tectal third was discarded, and anterior and posterior thirds were
homogenized separately in buffer (HB: 10 mM Tris
Cl, pH 7.4; 1.5 mM CaCl2)
with protease inhibitors (200 U/ml aprotinin, 50 µM leupeptin, 2 µM
pepstatin, 1 mM spermidine, and in some cases 50 µM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid;
Sigma, St. Louis, MO). Membranes were fractionated by centrifugation in
a sucrose gradient and washed and resuspended in PBS with
protease inhibitors (PBS+). The pellet was resuspended in PBS+ and
adjusted until a 1:15 dilution of the suspension in 2% SDS yielded an
optical density (OD) of 0.2 for anterior suspensions and 0.15-0.2 for posterior suspensions (results were similar with the two ODs) when
measured with 220 nm ultraviolet (UV) light. Latex microspheres that
fluoresce blue when exposed to UV illumination were added to the
posterior membrane suspension to reveal lane integrity. Alternating,
90-µm-wide anterior and posterior membrane lanes were laid down on a
Nuclepore filter (pore size 0.1 µm) by suction. E6 chick
embryos were used for the preparation of retinal strips. Retinas were
dissected from the sclera, and the pigment epithelium was removed. The
retina was cut into thirds parallel to the optic fissure. The central
third of the retina was discarded, and the temporal and nasal thirds
were mounted RGC side up on nitrocellulose paper (Sartorius) that had
been previously incubated in 0.01% Concanavalin A (Sigma) in L15
(Sigma) for 1 hr and thoroughly rinsed. To anterogradely label retinal
axon outgrowth in the standard branching assay and the cultures used
for time-lapse microscopy, retina was prelabeled with
4-(4-didecylaminostyryl)-N-methylpyridinium iodide
(4-Di-10-ASP; Molecular Probes) before explanting. For this
prelabeling, mounted retinal thirds were centrifuged for 10 min at 1200 rpm in 5 ml of a dye suspension (1:200 dilution in L15 of a 1% stock
solution of 4-Di-10-ASP in ethanol). Filter papers with retinas were
rinsed in L15 and incubated in medium (DMEM-F12 supplemented with 2 mM L-glutamine, 0.6%
D-glucose, 10 U/ml pen-strep, 20 mM HEPES, 5% rat serum or 2% chick serum, and
10% fetal calf serum) at 5% CO2 for 1 hr. The
temporal and nasal thirds of retina were cut into 300 µm strips on a
tissue chopper. The strips were placed RGC side down on the membrane carpets, parallel to the membrane lanes, which resulted in individual axons growing out from the explant crossing both types of lanes. Small
weights were placed on top of the ends of the strips to anchor the
explants and carpets. Two milliliters of medium, in some cases
supplemented with 0.4% methylcellulose, were added to each dish. For
the EphA3-Fc blocking experiments, 400 ng/ml of soluble rmEphA3-Fc (R & D Systems, Minneapolis, MN) or 400-800 ng/ml of human IgG, Fc portion
(Jackson ImmunoResearch, West Grove, PA) was added to the media.
In standard growth choice membrane stripe assays in which the retinal
explant is oriented perpendicular to the membrane lanes, 400 ng/ml of
EphA3-Fc was sufficient to eliminate the normal strong preference of
temporal axons to grow on anterior membranes (data not shown). Human-Fc
did not affect the growth preference of temporal axons at any
concentration tested (10-1500 ng/ml). Cultures were incubated in 5%
CO2 at 37°C.
Quantification of static cultures. After 48-72 hr of
incubation, standard branching cultures were fixed in 4% buffered
paraformaldehyde. In the EphA3-Fc and Fc experiments, axon labeling was
done by incubation for 5 min in 33 µM
carboxyfluorescein diacetate, succinimidyl ester in PBS (a fluorescent
vital dye; Molecular Probes), which labels all living cells and their
processes. Lanes and neurites were examined on an upright fluorescence
microscope (Nikon Microphot FX) and photographed with a 35 mm camera
using Ilford XP2 or Fujichrome film or imaged with a
silicon-intensified target (SIT) camera (Hamamatsu). Lanes were
visualized with UV illumination, and neurites with were visualized with
fluorescein illumination for anterograde DiAsp or vital dye
labeling, or rhodamine illumination for retrograde DiI labeling (see
below for labeling method).
We have used two methods for quantifying retinal axon branching:
anterograde and retrograde. All cases that met the following criteria
were analyzed: axons were well labeled, the membrane lanes were clearly
defined, and at least three axons or fascicles had grown across three
or more lanes. Branch counts were normalized for variations in lane
width (the first lane applied tended to be wider than the second lane).
Quantification was performed on photographs or video images of cultures
viewed with fluorescence illumination.
For anterograde quantification, instances in which an axon extended
from another axon at approximately a right angle and was not an obvious
instance of two axons intersecting were counted as branches.
Anterograde quantification has fewer complications than retrograde
quantitation, and therefore yields a higher number of cultures that can
be analyzed. However, it does not definitively distinguish between true
branches and abrupt, sharply angled deviations of axons previously
fasciculated. Retrograde quantification was used to label only true
branches. All cases used for anterograde quantitation of branching
preferences were also prepared for retrograde quantification. For this,
small deposits of a 2-5% solution of DiI in dimethylformamide (Sigma)
were placed distal to the explant in fixed cultures using a
Picospritzer. After 24-48 hr the dye had diffused throughout the axons
that contacted the injection site. Using this method, labeled neurites
proximal to the dye deposit but not extending into it can be
definitively identified as branches. Statistical significance of
branching preferences was assessed using the paired two-tailed,
Student's t test.
Time-lapse video microscopy. A proportion of the cultures
used for anterograde and retrograde analysis of axon branching were imaged with time-lapse video microscopy before fixation. Cultures were
imaged for 2-17 hr, beginning at 20-36 hr of incubation. For imaging,
the cultures were kept in an incubation box mounted on an upright
microscope (Nikon Microphot FX). The environment in the box was
maintained at 37°C in humidified 5% CO2 (95%
air). The microscope was equipped with a 100 W mercury epifluorescence light source, 10, 20, and 40× super-long working distance objectives (Nikon) and 20 and 40× water immersion objectives (Nikon), all with
high numerical apertures. Lanes were visualized with UV illumination, and the DiAsp-labeled axons were visualized with fluorescein
illumination. To avoid photo damage to fluorescently labeled
axons, video imaging was performed under low light level conditions,
and axons were exposed to light for very short periods spaced at
relatively long intervals. Neutral density filters were placed in the
light path to reduce the intensity of the fluorescent light. An
electronic shutter (Uniblitz) in the light path was controlled by
Image-1 software (Universal Imaging Corp.) to open for ~200 msec once every 2-5 min, during which four to eight images were taken with an
SIT camera and averaged. Focus was monitored to ensure that changes in
morphology were not the result of changes in focal plane.
Images were later transferred to an analog optical magnetic disc
recorder (Panasonic) to make "movies." Time-lapse image sequences were repeatedly played at various speeds ranging from 1 to 30 images
per second, analyzed for growth rate, branch extension, branch
retraction, growth cone bifurcation, and axonal deviations, and scored
according to the membrane substrate on which these events occurred.
Cultures in which axons appeared unhealthy, for example, axons that
developed a beaded appearance or exhibited widespread retraction, were
not analyzed. In all cases, branch counts were normalized for variable
lane width. Statistical significance of branching preferences was
assessed using the paired two-tailed, Student's t test.
Figures were prepared on a Macintosh computer using Adobe Photoshop,
Microsoft Excel, and Canvas software.
One potential concern with studies using time-lapse video microscopy is
the possibility that photo damage modifies axonal behavior such that
the analysis presents an incorrect representation of the actual events.
At variance with this possibility in our experiments is the finding of
no qualitative or quantitative difference in the branching preferences
exhibited by retinal axons in cultures that were not imaged compared
with those that were time-lapse video imaged before fixation and
further analysis. A frequently observed effect of over-illumination is
the cessation of axonal growth, growth cone exploration, and branching
activity, and in severe cases, axon beading and retraction. However,
with the precautions that we used, the axons and growth cones in our
cultures maintained their viability and motility. Even if a decrease in
motility or viability had occurred in our cultures, it would have had
little impact on the validity of our findings because the analysis of our time-lapse video images involved quantifying the relative frequency, rather than the absolute frequency, of events within each culture.
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RESULTS |
RGC axons overshoot their correct termination zone, but branching
along the axon shaft exhibits A-P topographic specificity
To investigate how RGC axons develop their topographic map along
the A-P axis of the tectum, small focal injections of DiI were made
into defined retinal locations during map development. We first
analyzed cases in which injections were made into peripheral temporal
retina, which maps to the anterior pole of the tectum, from E10, when
these axons initiate branching, to E13, when the topographic
organization of the projection can be identified (Nakamura and
O'Leary, 1989). Representative cases are illustrated in Figure 1. In these examples, as well as in all
cases analyzed, the location of the topographically correct TZ along
the A-P tectal axis was determined by mapping the injection site in the
retina onto the tectum (see Materials and Methods for details). The
labeling patterns reveal that the initial projection of temporal axons
to the tectum is topographically imprecise: at E10 most of the axons
project past the location of their topographically correct TZ in
anterior tectum. Some axons have branches, most of which extend at
right angles to the axon shaft. Interestingly, branch distribution is biased for the topographically correct location along the A-P axis. By
E11, branching is more pronounced, and some branches have begun to
develop immature arbors. The distribution of interstitial branches and
arbors becomes more restricted by E12 so that most are now located at
or near the correct TZ. By E13, most RGC axons establish projections to
the TZ through interstitial branches. Although many of the primary
axons still extend posterior to the TZ, the posterior extent of axon
overshoot is decreased.
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Figure 1.
Development of topographic projection from
peripheral temporal retina to anterior tectum. Confocal digital
montages of DiI-labeled RGC axons in tectal whole mounts from E10 to
E13. Axons were labeled by a small focal injection into peripheral
temporal retina ~1 d before fixation. Axons initially overshoot their
topographically correct TZ (center of future TZ, or TZ, is marked by
arrowheads) along the anterior
(A)-posterior (P) tectal
axis. However, branching along the axon shaft is biased for the
topographically correct location of the TZ at all ages. Between E10 and
E13, the topographic specificity of branch distribution increases, and
the extent of axon overshoot diminishes. Topographic connections to the
TZ are established by the arborization of topographically appropriate
branches. The anterior edge of the tectum is at the
bottom of each panel; only part of anterior tectum is
shown. The location of the topographically correct TZ along the A-P
tectal axis was determined, independent of the distribution of labeled
axons in the tectum, by mapping the injection site in the retina onto
the tectum (see Materials and Methods for details). Scale bar, 250 µm.
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To confirm our qualitative impressions of topographic map development,
we performed a quantitative analysis of 7-10 cases at each age (Fig.
2). We used cases in which 5-15 RGC
axons were well labeled, which allowed branching patterns of individual
axons to be resolved unambiguously. Each axon, along with its branches, was digitally traced to determine the distributions of axons, growth
cones, branches, and arbors relative to their predicted TZ (see
Materials and Methods for determination of predicted TZs, analysis
methods, definitions, and n values). The TZ is defined as a
zone extending 250 µm both anterior and posterior to a point along
the A-P tectal axis predicted by the location of the retinal injection
site and the mature topographic map. The 500 µm width of the TZ is
~5% of the A-P axis and corresponds to the approximate size of
typical mature retinal arborizations labeled by small focal DiI
injections in embryonic chick tectum (Thanos and Bonhoeffer, 1987;
Nakamura and O'Leary, 1989).
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Figure 2.
Development of topographic projection from
peripheral temporal retina to anterior tectum: quantitation of RGC axon
overshoot and branch distribution. Axons were labeled by a small focal
injection of DiI into peripheral temporal retina, as in Figure 1. The
anterior (A)-posterior (P)
tectal axis was divided into 500 µm bins; the number of labeled axons
and branches in each bin was counted, and the total number at each age
was summed. The x-axis plots the location of each 500 µm bin relative to the location of the topographically correct
termination zone (TZ) along the A-P tectal axis, which
on average was 1 mm from the anterior edge of the tectum.
A, Quantitation of RGC axon overshoot. Graphed are the
percentages of labeled axons that extend posteriorly past a given point
along the A-P tectal axis. B, Distribution of
interstitial branches along the axon shaft expressed in percentage. The
number of branches in each bin is graphed as the percentage of total
branches at a given age. C, Distribution of interstitial
branches along the axon shaft expressed as branch density. For each
age, the total number of branches in each 500 µm bin was divided by
total number of labeled axons within that bin to determine the number
of labeled branches per labeled axon per bin. Number of tectal whole
mounts: E10 (10), E11 (7), E12 (9), E13 (7). Number of axons:
E10 (104), E11 (64), E12 (68), E13 (52). Number of branches: E10 (302),
E11(248), E12 (241), E13 (141). See Results for statistical
tests.
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This quantitative analysis shows that essentially all RGC axons
overshoot the topographically appropriate location of their TZ along
the A-P axis. At E10, virtually all peripheral temporal axons extend
past their presumptive TZ, and more than half continue at least 1.5 mm
beyond it (Fig. 2A). These measurements probably underestimate the true degree of overshoot because some peripheral temporal axons are still growing posteriorly in the tectum at E10. Well
over 80% of RGC axons still extend past the TZ at E13, when the
location of the emerging TZ is clearly evident. However, the mean
overshoot decreases from 1.5 mm at E10 to 650 µm at E13 (p < 0.001; unpaired t test; E10:
n = 104, mean = 1496 ± 68 µm SEM; E13:
n = 52, mean = 658 ± 76 µm SEM). This
finding that RGC axonal growth cones initially grow well past their
correct TZ shows that the topographic organization of the chick
retinotectal projection is not developed by direct topographic growth
cone targeting.
In contrast to the lack of topographic growth cone targeting,
quantitative analysis shows that branch formation exhibits topographic specificity. At all ages from E10 to E13, branch distribution shows a
topographic bias for the correct A-P location of the future TZ in terms
of both overall branch distribution (Fig. 2B) and branch density (Fig. 2C). Although branch distribution and
density decline anterior and posterior to the appropriate site of the TZ, the decrease is asymmetric, with the slope of the decrease being
steeper anterior to the TZ than posterior to it. Both branch distribution and branch density increase in topographic specificity between E10 and E13, reflecting the maturation of the map. However, even at E10, the topographic bias in branch distribution is
statistically significant (p < 0.007;
 2 test; n = 302 branches).
Most axons, if not all, establish connections to the TZ through arbors
elaborated by branches rather than through a terminal arborization at
their leading growth cone (Figs. 1,
3A) (see Materials and Methods
for definitions of interstitial branch arbors and terminal arbors).
Figure 3A illustrates a typical case: interstitial branches
extend from the shaft of RGC axons millimeters behind their leading
growth cones and grow along the medial-lateral tectal axis to the
topographically appropriate TZ, where each branch forms a distinct
arbor within the TZ independent of the leading growth cone. At E10 and
E11, all axons that connect to the nascent TZ do so through an
arborization of their interstitial branches (n = 104 and 64 axons at E10 and E11, respectively) (Fig. 3B), and
all arbors in the TZ are formed by branches (Fig. 3C). At E12 and E13, most axons arborize in the TZ, and only a few axons have
the appearance of forming terminal arbors (Fig. 3B).
Consistent with this finding, >90% of arbors in the TZ are formed by
interstitial branches (p < 0.001 for all ages;
2 test; n = 26, 26, 74, and 75 arbors found in the TZ at E10, E11, E12, and E13, respectively)
(Fig. 3C). The true percentage of axons that form terminal
arbors in the TZ at E12 and E13 must be substantially lower than that
measured because changes in the positioning of interstitial branches
relative to the distal end of the primary axon, attributable to the
elimination of overshooting axon segments posterior to the TZ,
bring the distal end of many of the axons within 250 µm of the
TZ resulting in arbors that had initially developed as interstitial
arbors being redefined as terminal arbors (see Materials and Methods
for criteria).
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Figure 3.
Arbors in the termination zone are formed
by interstitial branches extended along the shafts of RGC axons.
Data presented in B and C were collected
from the same cases used in Figure 2. A, Arbor formation
by interstitial branches that extend from the shaft of primary RGC
axons to their termination zone (TZ). Shown is
fluorescence photomicrograph of RGC axons in an E12 tectal whole mount,
labeled by a small focal injection into peripheral temporal retina ~3
d before fixation. Arrows mark the branch points of four
interstitial branches extending from the shafts of two primary RGC
axons. Each branch extends medially along the medial-lateral tectal
axis and forms an immature arbor in the emerging TZ
(arrowhead). Anterior is to the bottom.
Scale bar, 100 µm. B, Percentage of RGC axons at E10
through E13 that arborize in their correct TZ by either the
arborization of an interstitial branch or by a terminal arborization
defined as an arbor formed at the distal end of the primary axon or by
a branch extended from the axon shaft within 250 µm of its distal
end. C, The percentage of arbors found within the TZ
that are formed by an interstitial branch or meet the criteria of a
terminal arbor. No terminal arbors are found at E10 and E11. The
percentage of terminal arbors at E12 and E13 is an over-representation
of the percentage that truly form as terminal arbors, because by these
ages the overshooting segments of axons distal to the TZ have begun to
be eliminated; thus, most if not all of the terminal arbors are
likely formed by interstitial branches that have come to be located
within 250 µm of the distal end of the retracting primary axon and
thus scored as terminal arbors. See Results for n
values and statistical tests.
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In conclusion, our quantitative data on the A-P distributions of growth
cones and interstitial branches, and the mode of arbor formation within
the TZ, strongly suggest that chick retinotectal topography is
established by the arborization of interstitial branches formed in a
topographic-specific manner along the axon shaft. A direct topographic
targeting and terminal arborization of the primary growth cone within
the TZ appears to play little, if any, direct role in topographic mapping.
Initial branch distribution is topographically specific regardless
of retinal origin of RGC axons
An analysis similar to that described above for peripheral
temporal axons was done at E10 and E11 to determine whether RGCs arising throughout the retina exhibit overshoot and
topographic-specific branching. At E10, we compared the distribution of
axons and branches labeled by small DiI injections made into peripheral
temporal and central retina (Fig. 4).
Nasal axons were not included because they have not yet extended far
enough to reach their topographically appropriate TZ in posterior
tectum. Both sets of labeled axons overshoot their topographically
appropriate TZ and both show biased distributions of branches along
their lengths. As described above, axons labeled from peripheral
temporal retina have a bias in branch distribution centered on the
topographically correct site of their future TZ in anterior tectum,
whereas the distribution of branches formed by axons labeled from
central retina is centered on the topographically correct site of their
future TZ in mid-tectum. Both axonal populations have a relative
paucity of branches anterior and posterior to their correct TZ (Fig.
4). At E11, axons arising from peripheral temporal, central, and
peripheral nasal retina all overshoot the topographically correct site
of their future TZ but exhibit a topographic bias in branch
distribution centered on their future TZ (Fig.
5A). Quantitation of branch
distribution confirms that branch number peaks at the location of the
topographically correct TZ and exhibits a sharp decline both anterior
and posterior to it (Fig. 5B). In conclusion, these findings
indicate that axons arising from all retinal regions overshoot their
topographically correct TZ but exhibit a topographic bias in branching
along the A-P tectal axis appropriate for the location of their future
TZ.
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Figure 4.
Initial branch distribution along the
anterior-posterior tectal axis is topographically specific regardless
of retinal origin of RGC axons. Shown are confocal digital montages of
RGC axons in tectal whole mounts labeled by a small focal DiI injection
into peripheral temporal retina or central retina 1 d before
fixation late on E10. Axons overshoot their topographically correct
termination zone (TZ; marked by
brackets), but the distribution of interstitial branches
along the axon shafts is strongly biased for the location of the future
TZ along the anterior (A)-posterior
(P) tectal axis at all ages. The location of the
topographically correct TZ along the A-P tectal axis was determined,
independent of the distribution of labeled axons in the tectum, by
mapping the injection site in the retina onto the tectum (see Materials
and Methods for details). The injection sites are plotted on drawings
of the retinal whole mounts and marked by arrows. Scale
bar, 500 µm. D, Dorsal; N, nasal;
T, temporal; V, ventral.
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Figure 5.
RGC axons overshoot their correct termination zone
in a position-dependent, differential manner. A,
Confocal digital montages of RGC axons in tectal whole mounts labeled
by a small focal DiI injection into peripheral temporal retina
(top), central retina (middle), or
peripheral nasal retina (bottom) 1 d before
fixation on E11. The injection sites are plotted on drawings of the
retinal whole mounts and marked by arrows. The relative
positioning of the labeled axons and branches within the tectum is
shown to the right with drawings of the outline of each
tectum on which the labeled axons and branches are traced. Axons
overshoot their topographically correct TZ (the predicted locations of
the TZs are marked with black arrowheads), but the
distribution of interstitial branches along the axon shafts
(white arrowheads) is strongly biased for the location
of the future TZ along the anterior
(A)-posterior (P) tectal
axis. Peripheral temporal axons exhibit the greatest overshoot and
peripheral nasal axons the least. The location of the topographically
correct TZ along the A-P tectal axis was determined, independent of the
distribution of labeled axons in the tectum, by mapping the injection
site in the retina onto the tectum (see Materials and Methods for
details). B, Distribution of interstitial branches along
the axon shaft expressed in percentage. The A-P tectal axis was divided
into 500 µm bins, and the number of branches in each bin is graphed
as the percentage of total branches for each of the three groups of
injections [number of cases quantified: temporal
(n = 10); central (n = 4);
nasal (n = 3); see Results for specific
n values and statistical tests]. To provide a more
direct comparison of relative developmental stages in axon branching,
branch (Figure legend continued.) (Figure legend continues.)
distributions were quantified at E10 for temporal injections and at E11
for central and nasal injections. Branch distribution is topographic
regardless of retinal origin. C, Average overshoot
measured for labeled axons per case relative to the location of the DiI
injection along the temporal-nasal axis of the retina. The extent of
RGC axon overshoot varies with retinal origin and shows a progressive
temporal to nasal decline in magnitude. At the ages analyzed, the A-P
axis of the tectum is ~10 mm at the center of its medial-lateral
extent. However, because of the shape and curvature of the tectum, some
views may give the impression that it is shorter. Scale bar (shown in
A): 500 µm for tectal montages of DiI-labeled axons
and 1100 µm for the drawings. D, Dorsal;
N, nasal; T, temporal; V,
ventral.
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RGC axons overshoot their correct termination zone in a
position-dependent manner
Over the course of analyzing the branching patterns of RGC axons
labeled from temporal, central, or nasal retina at E11, we noted that
the magnitude of axon overshoot of their TZ qualitatively appeared to
vary with retinal origin, with temporal axons exhibiting the greatest
overshoot of their topographically appropriate TZ and nasal axons
exhibiting the least (Fig. 5A). Analyses done at E10, E12,
and E13 give similar results (data not shown). To assess quantitatively
the relationship between the magnitude of overshoot and retinal
position, we measured at E11 the mean overshoot for RGC axons relative
to the retinal location of a focal DiI injection (Fig. 5C).
Peripheral temporal axons have a mean overshoot of 2 mm, which is
threefold greater than the 0.65 mm mean overshoot exhibited by
peripheral nasal axons. The mean overshoot progressively decreases as
the injection site moves from peripheral temporal to peripheral nasal
retina (r =  0.948; p < 0.0001;
correlation z test; n = 13) (Fig.
5C). These findings show that initial growth cone targeting
does exhibit a form of topography because, as a population, RGC axons
arising from different positions in the retina stop at different
locations in the tectum; however, these locations are substantially
posterior to the topographically appropriate TZs.
Refinement of the topographic map
Map refinement occurs through an increase in the topographic
specificity of interstitial branch and arbor distributions. Arbors become more restricted such that the percentage of interstitial arbors
located in the TZ increases from 55% at E10 to 93% at E13 (p < 0.001; 2
test; n = 55 and 77 arbors at E10 and E13,
respectively). The A-P extent of the branch distribution also becomes
more restricted to the topographically correct TZ: at E10, 32% of
branches are found in the TZ, compared with 58% at E13
(p < 0.001; 2
test; n = 302 and 141 branches at E10 and E13,
respectively) (Fig. 2B).
Examination of the mechanisms that underlie increased branch
specificity show that both branch addition and branch elimination are
differentially regulated over the length of the axon during map
refinement. From E10 to E13, branch density outside of the TZ decreases
by 38% from 0.51 to 0.35 branches per 500 µm segment of axon,
whereas branch density in the TZ increases by 58% from 0.92 to 1.58 branches (Fig. 2C). Elimination of the "overshooting" segments of the axon posterior to the TZ, and a relatively small percentage of branches formed along these segments, also contributes to
map refinement. However, because overall branch density posterior to
the TZ decreases substantially over this same period, the contribution of branch retraction independent of axon elimination to the increase in
topographic specificity in branch distribution is significant.
Mechanisms underlying map refinement can be further discerned by
comparing changes in the number and distribution of branches during the
remodeling process. From E10 to E11, mean number of branches per axon
increases by 35%, from 2.9 to 3.9 (p < 0.025; Mann-Whitney U test; n = 104 and 64 axons
at E10 and E11, respectively), then from E11 to E13 decreases to 2.7 branches per axon (p < 0.03; Mann-Whitney
U test; n = 64 and 52 axons at E11 and E13)
(Fig. 6A). Branch
density increases substantially in the TZ from E10 to E11, and the
relative increases in branch density at locations close to the TZ are
much greater than along the remainder of the axon. This suggests that
branches are preferentially added to more topographically correct
locations during this period, which increases overall specificity. In
contrast, most of the branches lost from E11 to E13 are eliminated from
locations outside the TZ, indicated by similar branch densities at E11,
E12, and E13 in the TZ and decreased branch density both anterior and
posterior to the TZ during this time. Preferential branch addition in
the TZ and branch elimination outside the TZ explain how the percentage of branches found in the TZ can increase by nearly
twofold from E10 to E13 (Fig. 2B), although the
average number of branches at E10 and E13 is virtually the same
(p > 0.9; Mann-Whitney U test;
n = 104 and 52 axons at E10 and E13) (Fig.
6A).
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Figure 6.
Refinement of the retinotectal map occurs through
topographic branch extension and ectopic branch elimination. Axons were
labeled by a small focal injection of DiI into peripheral temporal
retina ~1 d before fixation on E10-E13, as in Figure 1.
A, Average number of branches per axon. Branch addition
from E10 to E11 occurs primarily in the termination zone
(TZ), whereas branch elimination from E11 to E13 occurs
primarily outside of the TZ (refer to changes in branch density in Fig.
2C). B, Distribution of interstitial
branches according to length expressed in percentage. The anterior
(A)-posterior (P) tectal
axis was divided into 500 µm bins relative to the location of the
topographically correct TZ, and the number of branches in each bin is
graphed as the percentage of total branches of a given range of length.
Analyses were done at E12 and E13, and data were pooled. Longer
interstitial branches exhibit greater topographic specificity,
indicating that topographically appropriate branches are preferentially
extended. See Results for n values and
statistical tests.
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Further analysis indicates that branches positioned near the TZ
preferentially extend and arborize. At E12 and E13, branch distributions peak at the TZ regardless of branch length (Fig. 6B). However, longer branches show greater
topographic specificity than shorter branches. More than 60% of
branches 100-250 µm in length are found in the TZ, whereas branches
5-20 µm in length have a much broader distribution, with 33% found
in the TZ (p < 0.005;
2 test; n = 57 branches, 5-20 µm; n = 158 branches, 100-250 µm). Consistent with this finding, 55% of arbors compared with only 31% of
branches are found in the TZ at E10 (p < 0.015;
2 test; n = 302 branches and n = 53 arbors). By E13, >93% of all arbors are located in the TZ compared with 58% of branches
(p < 0.001; 2
test; n = 141 branches and n = 77 arbors). This suggests that initial branch formation is less
topographically specific than the subsequent arborization of these
branches and that branches located within the TZ exhibit a very
pronounced bias to extend and arborize at later ages. Thus, the
preferential extension and stabilization of appropriately positioned
branches appears to contribute to the increased specificity in the map
observed from E10 to E13.
Retinal axons exhibit topographic specificity in branching
in vitro
To investigate potential mechanisms that control the specificity
in RGC axon branching observed in vivo, we analyzed the
branching of chick RGC axons in vitro using a modified
version of the membrane stripe assay (Fig.
7). In this assay, explants of temporal
or nasal retina from E6 chicks are placed on a substrate of alternating lanes of membranes prepared from anterior or posterior tectum from
E9-E10 chicks (Fig. 7A-C). The axons extend
across the lanes and are labeled anterogradely with DiAsp (Fig.
7A,C) or retrogradely with DiI
(Fig. 7B). A larger data set can be collected with
anterograde labeling because of a higher success rate of labeling and
because the entire axonal population is labeled; however, in contrast to anterograde labeling, retrograde labeling unambiguously identifies true branches (Fig. 7D).
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Figure 7.
Chick temporal retinal axons exhibit topographic
specificity in branching in vitro. Anterograde and
retrograde axon labeling was used to assess the branching preferences
of temporal and nasal retinal axons extending across alternating lanes
of anterior and posterior tectal membranes. A,
Anterograde DiAsp labeling of temporal axons. Temporal axons form a
dense network of processes on the anterior membrane lanes
(A), oriented perpendicular to the primary axons
and indicative of branching, but not on posterior membrane lanes
(P). B, Retrograde DiI labeling of
temporal axons. A deposit of DiI (top) retrogradely
labels axons that contact it and all of their branches. Branch
formation by temporal axons is strongly biased for anterior membranes;
few branch points are found on posterior membranes. C,
Anterograde DiAsp labeling of nasal axons. Nasal axons branch profusely
but show no branching preference for either anterior or posterior
membranes. Scale bars, 100 µm. D, Quantification
scheme. Long axis of retinal explants (elongated ovals)
were parallel to tectal membrane lanes such that retinal axons would
extend perpendicular to the lanes. Circles mark
intersections between labeled processes that may be scored as branches.
Anterograde labeling: DiAsp was used to anterogradely label retinal
axons. Axons extending at approximately right angles from other axons
were scored as branches. Instances in which axons intersect and both
processes clearly extend beyond the intersection were not counted to
minimize the misidentification of defasciculating or crossing axons as
branches. Retrograde labeling: retrograde DiI labeling in fixed
cultures was used to identify branches unambiguously. Labeled processes
proximal to a DiI deposit (black
"cloud") but not in contact with it, and which
extend from DiI-labeled axons that do contact the deposit, were scored
as branches. Not all axons labeled by anterograde method are labeled by
the retrograde method. E, Quantification of branching of
temporal and nasal axons. Shown is the percentage of branches present
on anterior and posterior membranes; the number of branches on each
membrane type was normalized for lane width. Temporal axon branches are
preferentially found on anterior membranes. Branch distributions
obtained with anterograde (ant) and retrograde
(retro) labeling are similar. The distribution of nasal
axon branches does not have a bias for either set of membrane lanes.
The number of cultures of each type quantified is indicated.
F, The same data in E expressed as a
specificity coefficient [(number of branches on anterior
membranes number of branches on posterior membranes)/total
number of branches]. Positive coefficients of branching
indicate specificity for anterior membrane lanes; negative coefficients
indicate specificity for posterior membrane lanes. For example, a
coefficient of 1 indicates that all branches are on anterior membrane
lanes; 0 indicates an equal number of branches on each set of lanes.
See Results for n values and statistical
tests.
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Temporal axons show a strong bias in branch distribution, with most
branches found on anterior tectal membranes and few branches on
posterior tectal membranes, whether the axons are labeled anterogradely (Fig. 7A) or retrogradely (Fig. 7B). In contrast,
nasal axons do not exhibit a branching bias for either the anterior or
posterior membrane lanes (Fig. 7C). Quantification of the
anterogradely labeled cultures shows that 83 ± 1.5% of branches
along temporal axons are found on anterior membrane lanes
(n = 95 cultures, >2500 branches; p < 0.0001, Student's t test), whereas nasal axon branches are
equally distributed (Fig.
7E,F), with 53 ± 2.8%
formed on anterior membrane lanes (n = 14 cultures,
>400 branches; p < 0.0001). These data yield a
branching specificity coefficient of 0.67 ± 0.03 for temporal
axons and 0.06 ± 0.06 for nasal axons (Fig. 7F). Quantitation of the retrogradely labeled
cultures yielded branch distributions similar to those obtained with
anterograde labeling (Fig.
7E,F): 85 ± 2.6% of
temporal axon branches are found on anterior membrane lanes
(n = 15 cultures, >300 branches; p < 0.0001), and 57 ± 5.6% of nasal axon branches are found on anterior membrane lanes (n = 10 cultures, >200
branches; p < 0.0001). These data yield a branching
specificity coefficient of 0.70 ± 0.05 for temporal axons and
0.14 ± 0.11 for nasal axons (Fig. 7F). The
order in which the anterior and posterior membrane lanes were applied
to the filter was reversed in approximately half of the experiments and
found to have no significant reproducible effect on branch distribution
(data not shown). These findings demonstrate that branches formed by
temporal axons are preferentially distributed on anterior tectal
membranes, their topographically appropriate substrate, whereas nasal
axons show no significant preference.
Topographic branching in vitro is generated by
ephrin-A-mediated inhibition of branching
Our in vitro findings indicate that molecules
preferentially associated with either anterior or posterior tectal
membranes control the topographic branching of temporal axons. Because
ephrin-As, which are anchored to the cell membrane via a GPI-linkage,
are present at higher levels in posterior tectum than in anterior tectum and preferentially repel or collapse temporal RGC axon growth
cones, we suspected that the level of ephrin-As in posterior tectum can
inhibit branching along temporal axons. To test this idea, we performed
the membrane branching assay in the presence of soluble EphA3 receptor
bodies, which bind ephrin-As on tectal membranes and prevent EphA
receptors on retinal axons from encountering and being activated by
them (Marcus et al., 2000). Previous reports have shown the viability
of this approach in the membrane stripe assay (Ciossek et al., 1998).
For these assays, we added to the media either 400 ng/ml of a
recombinant mouse EphA3-Fc protein (EphA3 with the cytoplasmic domain
replaced by the Fc portion of human IgG) or 400-800 ng/ml of the Fc
portion of human IgG. In the standard membrane stripe assay used to
assess axonal growth preferences, the level of EphA3-Fc used eliminated
the normal strong preference of temporal axons to grow on anterior
tectal membranes caused by ephrin-A repellents on posterior
tectal membranes and had no effect on nasal axons (data not shown). The
human-Fc had no effect on the growth preferences of retinal axons at
any concentration examined, which ranged from 10 to 1500 ng/ml (data not shown).
In the presence of soluble EphA3-Fc, temporal axons do not exhibit
their normal strong preference to branch on anterior membranes and
instead branch equally on anterior and posterior membranes (Fig.
8A,C),
similar to nasal axons with or without the addition of soluble EphA3-Fc
(Fig. 8B,C). The branching
preferences of temporal and nasal axons in the presence of human-Fc is
the same as that observed on untreated tectal membranes (Fig.
8C). In the presence of Fc, 84 ± 1.9% of branches on
temporal axons are on anterior membranes (n = 6 cultures, >200 branches; p < 0.0001), with a
branching specificity coefficient of 0.69 ± 0.04 (Fig. 8C,D). However, in the presence of
EphA3-Fc, this preference was abolished, with 49 ± 2.0% of
branches on anterior membranes (n = 20 cultures, >1000
branches; p = 0.68; branching specificity coefficient
of 0.02 ± 0.04) (Fig. 8C,D). Nasal axons
did not show a preference for either set of membrane lanes in the
presence of Fc (51 ± 2.5% branches on anterior membranes;
n = 10 cultures, >750 branches; p = 0.82; branching specificity coefficient of 0.01 ± 0.05) or
EphA3-Fc (49 ± 1.6% branches on anterior membranes; n = 9 cultures, >700 branches; p = 0.51; branching specificity coefficient of 0.02 ± 0.03) (Fig.
8C,D).
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Figure 8.
Branching specificity of temporal axons on
anterior tectal membranes is attributable to ephrin-A inhibition of
branching on posterior tectal membranes. A,
B, Branching assays performed with EphA3-Fc added to the
media to assess the effect of blocking ephrin-A function on the
branching of temporal (A) or nasal
(B) retinal axons. As a control, human-Fc was
added to the media of similar cultures. In the presence of EphA3-Fc,
both temporal and nasal axons branch equally well on anterior
(A) and posterior (P)
tectal membranes. C, Quantitation of branching.
Percentage of branches formed on anterior and posterior membrane lanes,
normalized for lane width (number of cultures for each
condition noted above each set of bars). Temporal axons
show a branching preference for anterior membranes in control Fc
cultures but not in cultures containing EphA3-Fc. Nasal axons show no
branching bias in the presence of either control Fc or EphA3-Fc.
D, Specificity coefficients show that EphA3-Fc abolishes
temporal axon preference for branching on anterior membranes. See
Figure 7 legend for definitions and scoring criteria. See
Results for n values and statistical tests. Scale
bar, 100 µm. A3, EphA3-Fc in media; Fc,
human-Fc in media; TEMP, temporal.
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These findings indicate that the strong preference of temporal axons to
branch on anterior tectal membranes is caused by an ephrin-A-mediated
inhibition of branching on posterior tectal membranes. These findings
also suggest that the levels of ephrin-As present in posterior tectum
are sufficient to inhibit branching along temporal axons.
Dynamics of branching specificity revealed with time-lapse
video microscopy: modes of axon branching
To determine the mechanisms that lead to the strong bias in the
distribution of temporal axon branches in the branching assay, we used
low light level video microscopy to image over time living retinal
axons in approximately one-fourth of the cultures anterogradely labeled
with DiAsp. We were especially interested in determining the mode by
which branches form and whether the branching specificity observed in
fixed cultures is caused by preferential branch extension on anterior
membranes or by branch retraction on posterior membranes. True axon
branching can occur by the de novo formation of a branch along the axon shaft or by the bifurcation of the growth cone. In
addition, the sharp deviation of an axon from a fascicle can occasionally give the appearance of branching. Anterograde labeling of
fixed cultures does not distinguish between the true branching of axons
attributable to interstitial branching or growth cone bifurcation from
the appearance of branching attributable to a sharply angled deviation
of an axon from a fascicle, whereas retrograde labeling does not
distinguish between interstitial branching and growth cone bifurcation.
Time-lapse video microscopy shows that all three events occur in
vitro, with interstitial branching accounting for 22% of the
total number of "branches" scored, growth cone bifurcation accounting for 7%, and apparent deviations accounting for 71%. Examples of the branching of temporal axons on anterior membranes are
illustrated in Figures 9 and
10. Figure 9 shows an example of a
branch forming just behind the growth cone, an appearance that
resembles "backbranching" described by Harris et al. (1987) in frog
tectum (although in backbranching, the growth cone ceases its extension
and together with the backbranch forms a terminal arbor). Figure 10
shows an interstitial branch forming along the axon shaft well behind
the growth cone; this more closely resembles the branching phenomena
that we describe in vivo in chick tectum. Interstitial
branch formation occurs both while the growth cone of the primary axon
is actively extending over the membrane carpet and when it remains in
place. We did not observe a consistent correlation between a growth
cone contacting a lane border and the extension of an interstitial
branch behind it, nor with branching and the rate of growth cone
advance. Interstitial branches often form at the border between
anterior and posterior lanes, but they are also commonly observed to
form within an anterior lane well away from its borders. Figure
11A shows an example
of the branching of a temporal axon by growth cone bifurcation on an
anterior membrane lane: the growth cone of the elongating axon divides,
and each of the new tips extends as an independent axon collateral.
Analysis of time-lapse movies reveals that growth cones often advance
along previously established axons, and when they deviate at a sharp angle from the axon fascicle, the resultant static image (similar to
that obtained in the analysis of fixed cultures) in some instances can
have the appearance of a branch (Fig. 11B).
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Figure 9.
Branch forming on an anterior membrane lane a
short distance behind the growth cone of a chick temporal retinal axon
observed with time-lapse video microscopy. A,
B, Low-power view of axons (A) and
membrane lanes with posterior membrane lanes labeled with fluorescent
microspheres (B). The point of branching is
marked with an arrow in A, as well as in
C-G. C-G,
Formation of the branch over time. The branch is not apparent in
C but is visible 10 min later in D. Both
the branch (arrowhead in A and
E-G) and the main axon (small
arrow in A and
E-G) deviate and grow along the anterior
membrane lane (F, G). In this case, both
the branch and the main axon stop on the anterior membrane lane when
they reach its border with the posterior membrane lane; their growth
cones collapse, and they subsequently retract (data not shown). The
hours and minutes elapsed are noted on bottom right of
each panel. Scale bar (shown in A for
A and B): 50 µm; (shown in
G for C-G): 50 µm.
A, Anterior membrane lane; P, posterior
membrane lane.
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Figure 10.
Interstitial branching along the shaft of a chick
temporal retinal axon observed with time-lapse video microscopy.
Example of a branch extending from an axon shaft on an anterior
membrane lane. A, B, Low-power views of
axons (A) and lanes with posterior membrane lanes
labeled by fluorescent microspheres (B). The
arrow in A marks an interstitial branch.
C-F, High-power time-lapse views of the
de novo formation of the interstitial branch marked in
A; this branch forms well behind the leading growth
cone. The branch evident in D-F is not
present in C (arrow). The tension exerted
by the branch pulls the primary axon laterally. The hours and minutes
elapsed are noted on bottom left of panels. Scale bar
(shown in F): A, B,
100 µm; C-F, 50 µm.
A, Anterior membrane lane; P, posterior
membrane lane.
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Figure 11.
Growth cone bifurcation and axon deviations
observed with time-lapse microscopy. A, Retinal axon
branching attributable to growth cone bifurcation. The growth cone of
the axon (white arrow) bifurcates, forming two distinct
axon branches that diverge and extend. B, Deviation of
an axon from a fascicle. Fasciculated axons are present at time 0. Another axon has grown diagonally down from the top left
corner of the field (left-most arrow),
contacting an axon fascicle (right-most arrow). After
growing briefly down the fascicle, the axon deviates from fascicle
(middle arrow) and resumes growth on the membrane
substrate. The hours and minutes elapsed are noted on bottom
left of each panel. Scale bars, 25 µm.
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Time-lapse analysis of the generation of branching specificity
in vitro
Because anterograde quantification does not distinguish between
true branches and the appearance of branching by axon deviation from a
fascicle, the time-lapse equivalent to anterograde quantification is
the sum of all three types of events analyzed: interstitial branching,
growth cone bifurcations, and abrupt axon deviations. Analysis of
time-lapse videos of temporal axons reveals that all three events occur
predominantly on anterior membrane lanes, with a range of 86-94%
(number of branching events analyzed and statistical significance for
branching events on anterior versus posterior membranes: bifurcations, > 40, p < 0.0001; interstitial branching, > 125, p < 0.0005; deviations, > 400, p < 0.0001) (Fig. 12A).
When the data for the three events are summed for temporal axons, the percentage of events on anterior membrane lanes and the specificity coefficient (91%; p < 0.0001; specificity coefficient
0.82) (Fig. 12A,C) are similar to
those obtained with anterograde quantification of fixed cultures (Fig.
7E,F).
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Figure 12.
Branch extension and branch retraction contribute
to temporal retinal axon branching specificity. A,
Characterization of extension events seen with time-lapse imaging.
Percentage of events observed on anterior membranes and posterior
membranes. Interstitial branching (interst), growth cone
bifurcations (bifur), and deviations
(deviat) each occur more frequently on anterior
membranes. The combined total of these events
(total) is the time-lapse equivalent of the
branching data obtained with anterograde quantification of fixed
cultures. B, Percentage of branch extension
(ext; i.e., the combined total of interstitial branching
and growth cone bifurcation) and branch retraction (ret)
observed on anterior membranes and posterior membranes. The combined
total of interstitial branching and growth cone bifurcation is the
time-lapse equivalent of retrograde quantification of fixed cultures.
C, Specificity coefficients (number of events on
anterior lanes number of events on posterior lanes)/total
number of events) for the combined events "total" data in
A and the branch extension and retraction events data in
B. A coefficient of 1 indicates that the events occur
only on anterior lanes, and a coefficient of 0 indicates that they
occur equally often on anterior and posterior lanes. Because
retractions are regressive events, a negative coefficient of retraction
indicates a contribution to a positive branching coefficient. See
Results for n values and statistical tests.
A, Anterior; P, posterior.
|
|
The time-lapse equivalent of retrograde quantification of fixed
cultures is the sum of the true branching events, interstitial branching and growth cone bifurcation. Summing of these two events for
temporal axons yields a percentage on anterior membrane lanes and a
specificity coefficient (89%; p < 0.0001; specificity
coefficient 0.78) (Fig. 12B,C)
similar to the branching data obtained with retrograde quantification
of fixed cultures (Fig.
7E,F). These data indicate
that the bias in the distribution of temporal axon branches is caused
by the preferential extension of branches on topographically correct
anterior membranes. In conclusion, our time-lapse findings indicate
that temporal axons show a strong preference to branch on their
topographically appropriate tectal membranes.
Time-lapse video analysis reveals that branch retraction
(n > 100 retractions analyzed) contributes to
generating the biased distribution of temporal axon branches on
anterior membrane lanes observed in fixed cultures. Branches extended
by temporal axons are approximately twice as likely to retract on
posterior membrane lanes than on anterior membrane lanes (65% retract
from posterior membranes; p < 0.02) (Fig.
12B,C). However, for equivalent
time and fields of time-lapse analysis, branch extension is five times more frequent than branch retraction. These findings indicate that the
principal factor in establishing topographic specificity in branch
distribution exhibited by temporal axons in vitro is their
preferential extension of branches on anterior membrane lanes, although
a bias to retract branches from posterior membranes sharpens their
topographic specificity in branch distribution.
| |
DISCUSSION |
Figure 13 summarizes our in
vivo findings on the development of topography, which include the
following: (1) RGC axons overshoot the topographic location of their TZ
along the A-P tectal axis by a distance that varies with their origin
along the temporal-nasal retinal axis; (2) arbors are established by
branches that form along the axon shaft, and branches at the
appropriate A-P location preferentially arborize; and (3) axon
branching is topographically specific along the A-P axis, even at the
earliest stages that branches are detected. We show in vitro
that temporal axons extending across alternating lanes of anterior or
posterior tectal membranes preferentially branch on anterior membranes.
Use of EphA3-Fc to block ephrin-A function abolishes this branching
specificity and indicates that the level of ephrin-As in posterior
tectum is sufficient to inhibit temporal axon branching.
View larger version (31K):
[in this window]
[in a new window]
|
Figure 13.
Stages in the development of topographic
organization in the chick retinotectal projection. RGC axons initially
exhibit a position-dependent, differential overshoot of the topographic
location of their TZ along the anterior
(A)-posterior (P) tectal
axis: temporal axons overshoot the greatest distance and nasal axons
the least. In contrast, branches form along the shaft of RGC axons with
a substantial degree of topographic specificity for the A-P location of
their future TZ. Topography is enhanced through the preferential
arborization of appropriately positioned branches and elimination of
ectopic branches. N, Nasal; T,
temporal.
|
|
Our findings show that topographic branching along the shaft of RGC
axons is the critical event in developing the retinotectal map. To
date, the role of axon guidance molecules in RGC axon mapping has
focused on topographic growth cone targeting. However, the topographic
branching of RGC axons imposes different and more substantial
requirements on the molecular control of mapping than does growth cone
targeting and requires a reconsideration of mechanisms and the action
of ephrin-As in this process (Fig.
14).
View larger version (49K):
[in this window]
[in a new window]
|
Figure 14.
Actions and limitations of ephrin-As in
retinotectal map development and the hypothetical contributions of
other graded activities to generate topographic branching of RGC axons.
A, The top panel schematizes the
approximate gradient profiles for EphA receptors and ephrin-A ligands
in retina and tectum, respectively. The middle and
bottom panels illustrate the actions and limitation of
the graded ephrin-A repellents in topographic mapping, as indicated by
our findings. Temporal axons have higher levels of EphA receptors than
nasal axons; therefore, temporal axon growth cones will reach a level
of ephrin-A repellent signal sufficient to stop their advance anterior
to that for nasal axon growth cones. The nonlinear increasing ephrin-A
repellent gradient across the anterior
(A)-posterior (P) tectal
axis can account for the differential, position-dependent overshoot of
the termination zone (TZ) exhibited by RGC axons, which
is greatest for temporal axons and progressively declines for axons
originating from more nasal locations. Because the slope is shallow in
anterior tectum, temporal axons must extend farther past their future
TZ than nasal axons to achieve the same change in relative and absolute
levels of ephrin-As. In contrast, the ephrin-A repellent gradient alone
is insufficient to generate topographic branching along RGC axons. The
ephrin-As can inhibit branching along the segment of the overshooting
axons posterior to their correct TZ, but anterior to the correct TZ,
the level of ephrin-A repellent signal experienced by the axon shaft
would be below the threshold required to inhibit branching. Thus, if
only the tectal ephrin-As regulated branching, all RGC axons would
exhibit increased branching at more anterior positions in the tectum,
which have the lower levels of ephrin-A repellent signal.
B, C, Two potential models that can
account for topographic branching along RGC axons. Both models
incorporate the graded ephrin-A repellent and a distinct graded
activity that cooperates with it to generate topographic branching. In
each case, the ephrin-A repellent prevents branching along the axon
shaft posterior to the TZ, and the distinct graded activity regulates
branching along axons anterior to their TZ. The model in
B includes a distinct repellent in a gradient that
opposes the ephrin-A gradient and acts by inhibiting branching along
the axon shaft anterior to the TZ. Thus, branching along the axon shaft
occurs at an A-P tectal position below threshold for branch inhibition
for both of the repellent signals. The model in C
includes a branch-promoting activity in a gradient that parallels the
ephrin-A gradient. In this model, branching along the axon shaft occurs
at an A-P tectal position above threshold for the branch-promoting
signal but below threshold for branch inhibition by the ephrin-A
repellent signal. In each model, the position along the A-P tectal axis
at which an axon shaft exhibits preferential branching depends
on axon origin along the nasal-temporal retinal axis, which determines
the level of receptor expression for the two distinct activities.
N, Nasal; T, temporal.
|
|
Regulation of topographic branching through combinatorial
graded activities
The development of topographic retinotectal connections in frogs
(Holt, 1983, 1984; Sakaguchi and Murphey, 1985; Fujisawa, 1987) and
fish (Stuermer, 1988) occurs through the topographic targeting and
terminal arborization of RGC axon growth cones. In these species, RGC
growth cones do not overshoot their correct TZ but target it
appropriately and form terminal arbors in part through a process termed
backbranching. Backbranching was observed using time-lapse video
microscopy of developing retinotectal axons in Xenopus by
Harris et al. (1987), and subsequently by others in frog (O'Rourke et
al., 1994) and zebrafish (Kaethner and Stuermer, 1992), and is
characterized by the formation of short terminal branches at or near
the base of the leading growth cone as a mechanism used by RGC axons to
elaborate terminal arborizations in the tectum. Concurrent with
backbranching, the growth cone ceases its extension, often acquires a
branch-like morphology, and appears to collaborate with the
backbranches to form a terminal arbor. This phenomenon, as originally
defined, is clearly distinct from the interstitial branching that we
describe in the chick. In chick tectum, interstitial branches are found
along the shaft of RGC axons millimeters behind their growth cones, and
they often extend hundreds of micrometers along the medial-lateral
(dorsal-ventral) tectal axis before arborizing (present study)
(Nakamura and O'Leary, 1989; P. Yates and D. D. M. O'Leary,
unpublished observations); each branch forms its own distinct terminal
arbor and the leading growth cone does not participate in arborization.
Our observations strongly suggest that interstitial branches form along
the axon shaft hundreds of micrometers, even a millimeter or more,
behind the leading growth cone. For example, at E10 and E11, branches
are concentrated along axon shafts at the future TZ, 1-2 mm behind the
overshooting growth cones, and have the morphology of newly formed
branches, i.e., they are short and simple. In addition, the number of
branches per axon at the future TZ increases between E10 and E11,
whereas the axon overshoot also increases. These observations are
reminiscent of those made on the development of cortical layer 5 projections to the basilar pons in rodents. Static in vivo
observations of labeled layer 5 axons reveal short, simple branches
concentrated along the axon shaft above the basilar pons, ~4 mm
behind the leading growth cones (O'Leary and Terashima, 1988).
Time-lapse imaging of living hemibrain preparations definitively shows
that the corticopontine branches form de novo along the axon
shaft millimeters behind the advancing growth cone (Bastmeyer and
O'Leary, 1996).
Most models of retinotectal mapping have been based on the topographic
targeting and terminal arborization of RGC growth cones. This behavior
can be explained as a response to the increasing A-P gradient of
ephrin-A repellents: growth cones stop when they reach a threshold
level of repellent signal (Nakamoto et al., 1996). Because of their
higher level of EphA3, temporal axons are more sensitive to the
repellent than nasal axons and stop anterior to them. However, our
findings indicate that a principal role of ephrin-As in chick
retinotectal map development is to regulate topographic branching by
inhibiting branch formation along the overshooting segment of RGC axons
posterior to their TZ (Fig. 14A). However, the
ephrin-A repellent alone is insufficient to regulate branching, and
additional activities are required to prevent branching along the axon
anterior to the TZ.
Although many potential mechanisms could account for topographic
branching, Figure 14 illustrates two straightforward ones, each of
which include a graded activity that cooperates with the ephrin-A
repellent. This activity could be a branch-repellent gradient counter
to ephrin-As (Fig. 14B) or a branch-promoting gradient parallel to ephrin-As (Fig. 14C), with appropriate
receptor gradients in retina. The hypothetical counter-repellent could be mediated by ephrin-As and EphAs but expressed by RGCs and tectal cells, respectively. EphA3 is expressed in tectum in a decreasing A-P
gradient (Connor et al., 1998), and ephrin-A2 and ephrin-A5, which
appear to mediate bi-directional signaling after binding EphA3 (Huai
and Drescher, 2001), are expressed on RGC axons in an increasing
temporal-nasal gradient (Hornberger et al., 1999). In addition,
temporal axons expressing abnormally high levels of ephrin-A2 or
ephrin-A5 exhibit decreased branching and topographically aberrant and
diffuse projections within anterior tectum (Hornberger et al., 1999), a
phenotype consistent with axonal ephrin-As acting as receptors for an
EphA3 tectal repellent. Evidence consistent with a parallel
branch-promoting activity includes in vitro findings of
activities that promote nasal axon growth in the posterior part of
developing chick tectum (von Boxberg et al., 1993) or deafferented
adult rat SC (Bahr and Wizenmann, 1996).
Position-dependent overshoot exhibited by RGC axons
We show that RGC axons initially overshoot their TZ, indicating
that the level of repellent that growth cones encounter at the A-P
location of their future TZ is insufficient to stop their advance. In
contrast, our findings indicate that repellent levels insufficient to
stop growth cone advance are sufficient to prevent branching along the
axon. Thus, growth cone advance and interstitial axon branching appear
to exhibit different sensitivities to ephrin-A repellents. The
magnitude of overshoot is greatest for temporal axons and progressively
declines for axons from more nasal locations; this decline relates to
the slope of the combined A-P tectal gradients of ephrin-A2 and
ephrin-A5, which is shallow in anterior tectum and increases sharply
posteriorly (Monschau et al., 1997). Thus, temporal axons must extend
farther past their future TZ than nasal axons to achieve the same
change in relative and absolute levels of ephrin-As (Fig.
14A).
Chick temporal axons have been previously reported to overshoot their
TZ, but this was interpreted as a targeting error and not
representative of the population (Thanos and Bonhoeffer, 1987; Nakamura
and O'Leary, 1989). However, our findings indicate that the overshoot
is not an error but a normal response of RGC axons to guidance
molecules. The distance of overshoot along the A-P axis may be a
critical parameter influencing topographic branching along the axon.
For example, if receptors for molecules that influence branching are
graded along the shaft of an RGC axon, then the A-P location of
preferred branching along the axon would be affected by the distance of overshoot.
Topographic refinement of the retinotectal projection
We show that topographic specificity in branch distribution along
the A-P tectal axis increases with age, because of an increase in
branching near the TZ and a loss of branches outside it. In addition to
ephrin-As expressed by tectal cells, ephrin-As expressed on RGC axons
(Hornberger et al., 1999) may contribute to map development, especially
refinement (McLaughlin and O'Leary, 1999). As RGC axons arborize and
increase their surface area, the level of ephrin-As should also
increase. Because ephrin-A2 and ephrin-A5 are expressed in a high nasal
to low temporal gradient, the topographic arborization of nasal axons
should result in a substantial increase in ephrin-As in posterior
tectum. This increase in ephrin-As should decrease branch extension and
promote the elimination of branches and overshooting axons posterior to
their TZ. Thus, retinotectal map development may require the
contributions of ephrin-A repellents from both tectal cells and RGC
axons, which can explain why temporal axons establish permanent
arborizations in posterior SC in the absence of nasal axons, although
ephrin-A expression by collicular cells should be unaffected (Simon et
al., 1994). Patterned neural activity is also involved in map
refinement, because when activity is blocked a small proportion of
overshooting RGC axons persist and establish ectopic branches and
arbors well outside of their topographically correct TZ (Kobayashi et
al., 1990; Simon et al., 1992).
Species differences in development of topographic maps
Studies in frogs (O'Rourke and Fraser, 1994), fish (Kaethner and
Stuermer, 1992), chick (Thanos and Bonhoeffer, 1987; Nakamura and
O'Leary, 1989; present study), rat (Simon and O'Leary, 1992a,b), ferret (Chalupa et al., 1996; Chalupa and Snider, 1998), and wallaby (Ding and Marotte, 1997) indicate that topographic precision of initial RGC axon targeting in the tectum/SC differs across species. These differences can likely be accounted for by differences in the
expression of guidance molecules and the sensitivity of RGC axons to
them, including species differences in expression levels and patterns,
and family members expressed (Monschau et al., 1997; Connor et al.,
1998; Frisen et al., 1998; Vidovic et al., 1999; Brown et al., 2000;
Stubbs et al., 2000). For example, if the same concentration range of
ephrin-As is distributed along the A-P tectal axis in zebrafish as in
chick, the gradient slope would be much steeper in the smaller
zebrafish tectum. If the threshold of growth cone response to ephrin-A
repellents is conserved, a steeper gradient should result in enhanced
topographic precision in growth cone targeting, as observed in
zebrafish compared with chick. This proposal is supported by the
correlation between axon overshoot and the ephrin-A gradient in chick:
the greater overshoot by temporal axons than nasal axons correlates
with the shallow slope of the ephrin-A gradient in anterior tectum and
its steep slope in posterior tectum.
Growth cone targeting and axon branching are likely to be
controlled in part by the same topographic guidance molecules. If growth cone targeting is more precise, the initial topographic specificity in branching should be more precise. Studies and models of
retinotopic mapping should take both growth cone guidance and interstitial branching into account and attempt to provide a
parsimonious explanation for their molecular control.
| |
FOOTNOTES |
Received April 2, 2001; revised Aug. 10, 2001; accepted Aug. 15, 2001.
This work was supported by National Institutes of Health Grant EY07025.
We thank Glenn Friedman for contributing to the in vivo
analyses, and Octavio Choi and Geoff Goodhill for helpful comments on
this manuscript.
P.A.Y. and A.L.R. contributed equally to this work.
Correspondence should be addressed to Dennis D. M. O'Leary,
Molecular Neurobiology Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037. E-mail:
doleary{at}salk.edu.
| |
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Henke-Fahle S,
Bonhoeffer F
(1987b)
Avoidance of posterior tectal membranes by temporal retinal axons.
Development
101:909-913[Abstract/Free Full Text].
Copyright © 2001 Society for Neuroscience 0270-6474/01/21218548-16$05.00/0
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ABSTRACT
INTRODUCTION
