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First published online 24 July 2008
doi: 10.1242/dev.025049
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1 Program in Neuroscience, University of Utah Medical Center, 20 North 1900
East, Salt Lake City, UT 84132, USA.
2 Department of Neurobiology and Anatomy, University of Utah Medical Center, 20
North 1900 East, Salt Lake City, UT 84132, USA.
3 Brain Institute, University of Utah Medical Center, 20 North 1900 East, Salt
Lake City, UT 84132, USA.
Author for correspondence (e-mail:
chi-bin.chien{at}neuro.utah.edu)
Accepted 25 June 2008
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MATERIALS AND METHODS
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DISCUSSION
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Key words: Robo2, astray, ath5, atoh7, Morpholino, Fasciculation, Transplant, Cell-autonomy, Zebrafish
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; Guan and Rao,
2003; Tessier-Lavigne and
Goodman, 1996). The growth cone has two potential sources of
guidance information. First, it may fasciculate with `pioneer' axons - defined
here as earlier-born axons that share its path - using cell-adhesion
molecules. Second, it may sense secreted or cell-surface guidance ligands
produced by other cells in the brain, acting at long or short range,
respectively. Most recent work has focused on guidance ligands; genetic
screens and biochemical purification strategies have identified many
ligand/receptor pairs used by navigating growth cones
(Dickson, 2002;
Guan and Rao, 2003;
Tessier-Lavigne and Goodman,
1996).
There is long-standing evidence, however, that pioneer-follower
interactions can play necessary roles in guidance
(Lopresti et al., 1973;
Bate, 1976;
Raper et al., 1983;
Raper et al., 1984;
Kuwada, 1986;
Klose and Bentley, 1989;
Ghosh et al., 1990;
Pike et al., 1992;
Hidalgo and Brand, 1997;
Jhaveri and Rodrigues, 2002;
Williams and Shepherd, 2002).
In other cases, pioneers are not required
(Keshishian and Bentley, 1983;
Holt, 1984;
Eisen et al., 1989;
Cornel and Holt, 1992) or only
facilitate followers' guidance (Chitnis and
Kuwada, 1991; Pike et al.,
1992; Bak and Fraser,
2003). Nearly all of these studies were carried out in simple
systems where a few pioneer axons could be identified, and where required
roles for pioneers were tested by ablation. Furthermore, these pioneers were
usually heterotypic: of a different neuronal type than the followers, with
different origins and targets, so that they could only guide the followers
through one leg of their journey. Thus, it is not clear (1) whether the role
of pioneers is generalizable to more complex systems; (2) what the sufficient
functions of pioneers are (for instance if they take abnormal paths); or (3)
what roles are usually played by isotypic interactions (between axons from the
same neuronal type).
In vertebrates, most axon tracts are built on a large scale. They typically
comprise thousands of axons, all with the same origin and target, and often
develop over an extended period as new neurons are added. This raises the
possibility that vertebrate axons could be guided by isotypic interactions,
either between earlier and later axons (pioneer-follower interactions), or
between axons that grow at the same time (community interactions). As axons in
the same tract share both origin and target, isotypic interactions could act
at multiple choice points throughout their pathway. Although there is often an
unspoken assumption that vertebrate axons indeed use pioneers for guidance,
there have been only a few direct experimental tests of this hypothesis
(Holt, 1984;
Eisen et al., 1989;
Ghosh et al., 1990;
Cornel and Holt, 1992;
Bak and Fraser, 2003). Here, we
have used the zebrafish retinotectal system to study this fundamental cellular
mechanism in axon guidance. We first ask whether isotypic pioneer-follower and
community interactions are important in the development of a large vertebrate
axon tract. We next use a pioneer replacement strategy to test whether
pioneers are sufficient to affect followers, and to assess the relative
importance of axon-axon interactions compared with guidance receptor
signaling.
The retinotectal projection is one of the best-studied vertebrate tracts,
the formation of which has been studied extensively
(Erskine and Herrera, 2007).
Retinal ganglion cell (RGC) axons must navigate out of the eye, across the
optic chiasm, and dorsally through the optic tract to reach the optic tectum.
Although retinal axons parallel the tract of the postoptic commissure (tPOC)
after crossing the optic chiasm, they make at most fleeting contacts with tPOC
axons (Burrill and Easter,
1995), and embryological manipulations that remove tPOC axons do
not affect retinal axon guidance (Cornel
and Holt, 1992). Thus, retinal axons do not require heterotypic
pioneers. Despite the extensive retinotectal literature, there has been only
one functional test of whether retinal pioneer axons might guide later retinal
axons (i.e. through an isotypic interaction). In Xenopus,
dorsocentral RGCs are the first to send out their axons. There was no effect
on the guidance of later retinal axons when heterochronic transplants were
used to delay the outgrowth of dorsocentral RGCs
(Holt, 1984), suggesting that
retinal pioneers are not required. Here, we re-examine the role of retinal
pioneers in guidance both within the eye and after exiting it. We use genetic
and embryological manipulations both to remove early RGCs, and to replace them
with cells that lack the Robo2 guidance receptor. We find that isotypic
pioneers in fact play multiple roles during the formation of this archetypal
vertebrate tract.
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), and the
null ast allele astti272z
(Fricke et al., 2001). As
ast is homozygous adult viable, ast embryos were generated
by incrossing homozygote parents. Embryos were raised at 28.5°C in 0.1 mM
phenylthiourea and staged according to time postfertilization and morphology
(Kimmel et al., 1995).
Experimental procedures followed NIH guidelines and were approved by the
University of Utah Institutional Animal Care and Use Committee.
Morpholino injections
Lyophilized ath5MO
(5'-TTCATGGCTCTTCAAAAAAGTCTCC-3', antisense start codon
underlined; Open Biosystems/Gene Tools) was solubilized in 1x Danieau's
buffer and aliquotted at -20°C. Embryos from either isl2b:GFP/+
or isl2b:mCherry-CAAX/+ incrosses were collected at the one-cell
stage, and a nominal volume of 1 nl MO was pressure injected at the yolk/cell
interface using a Picospritzer (Parker). MO was diluted to working
concentrations with 0.1% Phenol Red as a marker dye, and the injected bolus
measured using an eyepiece micrometer. All embryos for the dose-response
experiments of Figs 1 and
2 were injected in the same
session with a single pipette, counting pressure pulses to deliver different
doses. Individual live embryos were repeatedly assayed for GFP expression in
the retina using a fluorescent dissecting microscope, every 3 hours from 33-57
hpf. Repeating this experiment yielded essentially the same results, except
for a shift attributable to different bolus size (data not shown).
Cell transplants
Transplants were performed as described by Ho and Kane
(1990). Briefly, one-cell
donor embryos were injected with 5% rhodamine dextran (10,000 MW) as a lineage
marker; at 4 hpf, 20-50 cells were transplanted to the animal pole of host
embryos, which were raised to 5 dpf at 28.5°C. For
Fig. 4, donors were from an
isl2b:mCherryCAAX/+ incross, while hosts were isl2b:GFP
embryos injected with 5 ng ath5MO.
In addition to RGCs, isl2b:gfp labels small clusters of neurons in
the forebrain and dorsal diencephalon that make it difficult to unambiguously
identify misrouted retinal axons. Therefore, axon-axon interaction experiments
used the Tg(brn3c:gap43-GFP)s356t transgene, which labels
a subpopulation of 
50% of RGCs without confounding brain expression
(Xiao et al., 2005). For
Fig. 5, donors were from a
brn3c:GFP/+ or ast; brn3c:GFP/+ incross, whereas hosts were
wild type or ast. For Fig.
6, donors were nontransgenic, while hosts were brn3c:GFP
embryos injected with 5 ng ath5MO.
Immunofluorescence and staining
For whole-mount immunostaining, isl2b:GFP-positive larvae were
fixed at 5 or 6 dpf in 4% paraformaldehyde (PFA) in PBS overnight at 4°C,
washed in PBST (PBS with 0.1% Tween-20), dehydrated through a methanol series,
stored at -20°C for over 12 hours, rehydrated, washed in PBST,
permeabilized with 0.1% collagenase for 1 hour at room temperature, then
incubated in the following primary antibodies overnight at 4°C: rabbit
anti-GFP (1:400; Invitrogen A11122), mouse anti-GFP (1:200; Chemicon MAB3580),
rabbit anti-DsRed (1:200; Clontech 632496), mouse anti-parvalbumin (1:400;
Chemicon MAB1572) or affinity-purified rabbit anti-Pax2a (1:300; gift of A.
Picker). Larvae were then washed in PBST, incubated in goat anti-rabbit Alexa
488 (1:200; Invitrogen A11008), goat anti-mouse Cy3 (1:200; Jackson
ImmunoResearch 115-165-003), goat anti-mouse Alexa 488 (1:200; Molecular
Probes A-11029) or goat anti-rabbit Cy3 (1:200; Jackson ImmunoResearch
111-165-003), then stained in Hoechst 33342 (1:15,000; Molecular Probes
H-3570) or ToPro-3 (1:1000; Molecular Probes T3605) for 30 minutes at room
temperature. Whole-mount in situ hybridization for netrin1a was
performed as described previously (Wilson
et al., 2006). For sectioning, embryos were dehydrated in
methanol, infiltrated at 4°C in 1:1 Immuno-Bed:methanol for 30 minutes
then 100% Immuno-Bed overnight, oriented and embedded in 20:1
Immuno-Bed:Immuno-Bed Solution B (EMS 14260-04), and sectioned at 8 µm on a
Reichert-Jung 2050 Supercut microtome with a glass knife.
Fluorescent microscopy
GFP expression was initially assayed using an Olympus SZX-12 fluorescent
dissecting microscope with a 1.6x objective. For more detailed analysis
of GFP expression and the retinotectal projection, live 5 dpf larvae were
mounted in 1.5% low-melt agarose with tricaine and imaged with an Olympus
confocal microscope. Images were processed using Adobe Photoshop CS2. The
relatively small number of GFP+ axons in cell transplant experiments are
obscured by skin autofluorescence when viewed as confocal projections.
Therefore, for Figs 5 and
6, we used ImageJ
(http://rsb.info.nih.gov/ij;
developed by Wayne Rasband, NIH) and Photoshop to manually edit each z-slice
and remove autofluorescence and background fluorescence, taking care always to
spare nearby axons (see Fig. S3 in the supplementary material). Raw confocal
stack data is available upon request. For cell counting, 42 hpf
isl2b:gfp eyes were fixed, labeled with ToPro-3, dissected, mounted
in 80% glycerol and imaged with an Olympus confocal microscope. ToPro3+, GFP+
nuclei were counted by manual inspection of each z-slice in the
stack, and movies were made with Volocity software.
Phenotype quantification
In ast, eight retinal pathfinding errors are commonly seen:
midline crossing in the habenular and posterior commissures, and left- and
right-sided projections to the telencephalon, diencephalon and ventral
hindbrain (see Fig. S2 in the supplementary material). For each chimeric
larva, the presence (one or more axons) or absence of each error was scored at
5-6 dpf by an observer blinded to genotypes, using ImageJ to examine the
entire (unedited) confocal z-stack. Scores were compared by the
Mann-Whitney U test using Instat 3 (GraphPad).
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40 hpf (Hu and
Easter, 1999; Masai et al.,
2005). After this, new RGCs are added in successive peripheral
rings (Fig. 1A). The
RGC-specific transgene Tg(isl2b:GFP)zc7 turns on by 30 hpf
in ventroanterior retina, reliably labeling all or the vast majority of RGCs
(Fig. 1B; A.J.P. and C.-B.C.,
unpublished). To remove early RGCs, we used a translation-blocking antisense
morpholino oligonucleotide (ath5MO) to knock down function of
ath5 (atoh7 - Zebrafish Information Network), a bHLH
transcription factor expressed specifically in the eye and required
cell-autonomously for RGC differentiation
(Kay et al., 2005).
ath5 mutants completely lack RGCs
(Brown et al., 2001;
Kay et al., 2001), and their
RGC progenitors seem instead to take on primarily bipolar cell fates
(Kay et al., 2001). We
reasoned that ath5MO should have a similar, but transient, effect as
the efficacy of morpholinos wanes as their concentration is diluted by
embryonic growth (Nasevicius and Ekker,
2000). Indeed, we found that ath5MO causes a loss of
early-born central RGCs, but allows later-born peripheral RGCs to
differentiate (Fig. 1C). In
isl2b:GFP embryos injected with high-dose (5 ng) ath5MO
(Fig. 1C;
Fig. 2E; see Fig. S1 in the
supplementary material), RGCs were found exclusively in peripheral retina at 6
days postfertilization (dpf) - over 4 days after central RGCs normally form -
showing that differentiation is prevented rather than merely delayed. We next
injected an ath5MO dose series, monitoring RGC differentiation in
live embryos from 33 to 57 hpf. In control embryos, GFP+ RGCs were always
present by 33 hpf. With successively higher doses, the first RGCs appeared
successively later, until with 5 ng ath5MO RGCs did not appear until
42 hpf or later in 90% of embryos (Fig.
1D). Thus, varying the dose of ath5MO controls the time
at which the first RGCs differentiate.
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), and
for netrin1a, a marker of the optic nerve and optic nerve head
(Fig. 3). Both markers appeared
normal in ath5 morphants. Taken together, these results strongly
suggest that an isotypic pioneer-follower interaction is required for the
axons of later RGCs to exit the eye, and, further, that this interaction
occurs during a critical period before 42 hpf.
We next tested whether this pioneer-follower interaction is sufficient as
well as necessary, by resupplying RGCs to ath5 morphants. We targeted
the presumptive eye field in blastula-stage transplants from
isl2b:mCherry-CAAX donors into isl2b:GFP hosts injected with
high-dose ath5MO (Fig.
4A). As ath5 acts cell-autonomously
(Kay et al., 2005), we
expected that donor (non-morphant) RGCs would differentiate without affecting
host cells. Indeed, whereas host RGCs were still restricted to peripheral
retina, donor RGCs were found in central retina, and usually sent axons out of
the eye to the tectum (Fig.
4B,C). Of 32 eyes with mCherry+ donor axons reaching the tectum,
24 (75%) showed rescue of retinal exit, with GFP+ host axons reaching the
tectum (Fig. 4B). Retinotectal
topography appeared unaffected: donor axons from central retina projected to
central tectum, and host axons from peripheral retina projected to peripheral
tectum (Fig. 4B). Furthermore,
imaging within chimeric eyes showed that late-born host axons appeared to
fasciculate with donor axons (Fig.
4C-C''). Therefore, early RGCs are both necessary and
sufficient to guide later axons out of the eye.
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). Transplantation of eye primordia yields strictly
eye-autonomous pathfinding defects, showing that Robo2 is required in the eye
and not the brain (Fricke et al.,
2001). Here, we performed cell transplants from WT or ast
donors labeled with the RGC-specific transgene brn3c:GFP
(Xiao et al., 2005) into
nontransgenic wild-type or ast hosts
(Fig. 5A). We then assayed the
pathfinding of GFP-positive axons (which bear the donor genotype),
quantitating the results by counting the number of classes of pathfinding
errors made by GFP-positive donor retinal axons, yielding a score for each
host from 0 for wild type to 8 for strong ast (see Fig. S2 in the
supplementary material; see Materials and methods).
As expected, wild-type donor axons in wild-type hosts made no pathfinding
errors (Fig. 5B,B';
0.0±0.0 errors, mean±s.e.m.). Surprisingly, wild-type axons
transplanted to ast hosts always made errors, which occurred at
several different positions (Fig.
5C,C'; 2.71±0.40 errors,
P<0.0001). Presumably, despite expressing functional Robo2,
they were misguided by neighbors that lacked Robo2. Donor ast axons
made many errors in ast hosts
(Fig. 5D,D';
7.93±0.07 errors), similar to axons in nontransplanted ast
embryos (see Fig. S2 in the supplementary material). By contrast, ast
axons transplanted into wild-type embryos navigated far better
(Fig. 5E,E';
2.86±0.31 errors, P<0.0001). Thus, even lacking functional
Robo2, their pathfinding behavior was significantly rescued by wild-type
neighbors. These effects are not transgene-dependent: using isl2b:GFP
instead of brn3c:GFP yielded qualitatively similar results (data not
shown). Strikingly, then, Robo2 acts nonautonomously in cell transplants,
unlike its autonomous behavior in eye transplants
(Fricke et al., 2001). As a
receptor, the direct effects of Robo2 must take place in the growth cone that
expresses it, but this does not mean that indirect effects cannot affect other
axons. The strong effect of the genotype of the host axons on donor axon
behavior shows that, despite the clear importance of Slit-Robo signaling, its
role in individual axons can be overridden by axon-axon interactions.
Is this a pioneer-follower effect? As these chimerae contained far more host than donor cells, the host genotype could be affecting donor axon behavior because most pioneer axons are likely to be host derived. If so, replacing pioneer RGCs with cells of a different genotype should alter pathfinding by later axons. We tested this prediction by injecting high-dose ath5MO into brn3c:GFP hosts to remove all early RGCs, replacing the pioneer RGCs using transplants from nontransgenic donor embryos, then at 5 dpf assaying the pathfinding of GFP-positive axons, which are host-derived and therefore `follower' axons (Fig. 6A). Wild-type followers made no errors when transplanted pioneers were wild type (Fig. 6B,B'; 0.0±0.0 errors). However, when pioneers were replaced with ast cells, wild-type followers made more errors (Fig. 6C,C'; 1.72±0.31 errors, P<0.002). Thus, misguided pioneers indeed influence wild-type followers. As expected, ast followers made many errors when pioneers were ast (Fig. 6D,D'; 5.96±0.33 errors). When pioneers were replaced with wild-type cells, ast follower axons made fewer errors (Fig. 6E,E'; 4.79±0.24 errors, P<0.01). Thus, the pathfinding of follower axons is partially influenced by the genotype of the pioneers, although clearly the genotype of the followers remains the predominant influence.
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), which did not
find an important role for retinal pioneers. We believe the difference derives
from our ability to use ath5MO injection to completely prevent the
development of all central RGCs. By contrast, the heterochronic transplants
used in the Xenopus study only affected the dorsal half of central
retina (the source of pioneers in Xenopus), and furthermore only
delayed the development of retinal pioneers by 9-20 hours, rather than
removing them entirely. A previous study in zebrafish had proposed that a
small group (<10) of early retinal growth cones were pioneers based on
their significant separation from the next growth cones to follow
(Stuermer, 1988). It is clear
from our ath5 morpholino injections that these few growth cones are
not functionally pioneers, in the sense of being necessary for the guidance of
later axons. Instead, we find that far more cells must be removed to prevent
retinal exit: the RGCs born by 42 hpf, the critical stage
(Fig. 1), number about 600 (see
Movies 2 and 3 in the supplementary material). This is strikingly different
from invertebrate systems where ablation of a few pioneers is sufficient to
perturb follower axons.
After retinal exit, our transplant experiments show that isotypic
interactions between retinal axons are important for three further choice
points that require Robo2 function (Fricke
et al., 2001; Hutson and
Chien, 2002) (M. Hardy and C.-B.C., unpublished). Misrouting in
the optic chiasm leads to chiasm defasciculation
(Fig. 6C); misrouting in the
ventral optic tract leads to telencephalic or ventral hindbrain projections
(Fig. 5C;
Fig. 6C); and misrouting in the
dorsal optic tract leads to aberrant crossing in the habenular or posterior
commissures (Fig. 5C). At all
three choice points, mutant axons can lead wild-type neighbors into error.
Thus, axon-axon interactions function throughout the entire course of the
retinotectal projection.
Several questions remain for future studies on axon-axon interactions. What
distinguishes pioneers from followers? We know of no molecular markers that
distinguish these RGC populations - for instance, they both express
robo2 (Campbell et al.,
2007) - so the only difference may be the time and position of
their birth. How do pioneers interact with followers within the eye? Most
simply, later axons may fasciculate with early axons, as seen in
Fig. 4C-C''; future
experiments will test whether disrupting cell-adhesion molecules leads to axon
guidance errors within the retina, as in other vertebrates
(Brittis et al., 1995;
Leppert et al., 1999;
Ott et al., 1998;
Zelina et al., 2005). However,
we cannot formally exclude other possibilities. For example, early RGC cell
bodies might secrete an attractant that draws later axons to the optic nerve
head. Interestingly, WT>ath5 morphant transplants in which host
axons remained trapped within the eye tended to have fewer donor axons on the
tectum (data not shown), presumably reflecting fewer donor RGCs, which might
provide an insufficient level of attraction. What underlies the apparent
crucial period before 42 hpf? Both timing and spacing are possible
explanations. Ligands implicated in retinal exit
(Birgbauer et al., 2000;
Deiner et al., 1997;
Kay et al., 2005;
Kolpak et al., 2005;
Li et al., 2005;
Thompson et al., 2006) or
their receptors, might be expressed only transiently during this period, so
that RGCs born after 42 hpf would lack appropriate exit signals.
Alternatively, early-born RGCs may be close enough to find the optic nerve
head, whereas later-born RGCs are simply too far away to sense it.
Although most pioneer experiments have studied heterotypic axon
interactions, there have been a few studies of isotypic interactions. In
grasshopper, Myers and Bastiani (Myers and
Bastiani, 1993) found that the growth cone of the identified Q1
neuron interacts strongly with its contralateral homolog, and that ablation of
one Q1 often leads to midline stalling of the growth cone of the other Q1. In
the zebrafish, Bak and Fraser (Bak and
Fraser, 2003) found that pioneer and follower growth cones in the
postoptic commissure (POC) display characteristic morphology and kinetics
(spread/slow and narrow/fast, respectively). After laser ablation of pioneer
growth cones, followers appeared to take their place and behave like pioneers.
However, POC pioneer ablation did not have any effect on the pathfinding of
follower axons, and indeed we do not know of previous studies in vertebrates
showing guidance by isotypic pioneers.
Here, we found that isotypic interactions after retinal exit help to guide
retinal axons to the tectum. We were able to test the role of axon-axon
interactions in a new way: by replacing rather than ablating RGCs, we tested
sufficiency rather than necessity. Previous studies on pioneers found that
their ablation prevents normal pathfinding by followers (e.g.
Raper et al., 1983;
Klose and Bentley, 1989;
Pike et al., 1992;
Whitlock and Westerfield,
1998); here, we used zebrafish transplants to test how misrouted
mutant axons affect wild-type axons, and vice versa. We found that
ast host axons can misroute wild-type donor axons, whereas wild-type
host axons rescue ast donors to a large degree. This allowed us to
compare the relative importance of different guidance mechanisms; we conclude
that axon-axon interactions can be just as important as cell-autonomous
Slit-Robo signaling. As well as showing a significant role for
pioneer-follower interactions, our data suggest an even more important role
for peer interactions between late retinal axons. An ast host axon in
a WT>ast;ath5MO transplant
(Fig. 6E,E') misroutes
more often than an ast donor axon in an ast>WT transplant
(Fig. 5E,E'), perhaps
because a larger fraction of RGCs are ast in the former case. What
might distinguish pioneer-follower from peer-peer interactions? We do not
necessarily expect different molecular interactions but, instead, differences
in temporal and spatial proximity. Peers grow out at the same time, whereas
peripheral followers grow out significantly later than pioneers. New retinal
axons grow at the surface of the brain, so that pioneers are gradually buried
underneath. Thus, although peers can interact directly, late followers will
contact pioneers only indirectly, with several degrees of separation.
Our results are complemented by a recent study
(Gosse et al., 2008), which
showed that even a single RGC transplanted into lakritz/ath5 mutant
hosts can sometimes navigate successfully to the tectum. Successful cases of
tectal innervation showed a bias for central RGCs; furthermore, axons of
single RGCs transplanted to peripheral retina often remained trapped within
the eye. Tectal innervation appeared to be a rare event, as a large number
(5000) of transplants had to be performed; in similar experiments,
transplanting a few RGCs into lakritz or ath5 morphants, we
also find that axons rarely reach the tectum (A.J.P. and C.-B.C.,
unpublished). Overall, these data are consistent with our model that a large
population of central RGCs are required for peripheral axons to exit the
retina.
Our transplant paradigms create artificial situations in which axon-axon interactions are at war with ligand-receptor signals. For instance, a wild-type axon surrounded by ast axons may be shown the correct path by signals from the brain, but tugged off-course by its neighbors. During normal retinotectal development, by contrast, all axons are wild type. Not only does every axon recognize the correct path to its target, but so do all its predecessors and neighbors. Therefore, signals from both outside the tract (guidance ligands) and within (axon-axon interactions) should act in coordination to produce the highly stereotyped, precise formation of this vertebrate axon tract. As interaction (fasciculation) between isotypic axons is widely observed, we propose that this coordinated strategy is probably used throughout the development of complex vertebrate nervous systems.
Finally, we point out significant implications for the interpretation of mutant axon guidance phenotypes. For example, imagine a tract that uses two guidance receptors, A and B, to sense partially redundant sets of guidance signals. Suppose that in the absence of fasciculation, knockout of A would cause 30% of the axons to misroute, whereas knockout of B would cause 10% errors. As most axons still navigate correctly, axon-axon interactions might in fact reduce the A mutant phenotype to 10%, and the B mutant phenotype to undetectable levels. Thus, genetic redundancy may act not only at the level of single axons, but also at the level of entire tracts.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/17/2865/DC1
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DISCUSSION
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Y., Baier, H., Nonet, M. L. and Chien, C. B. (2007). Slit1a
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