Morpholinos for splice modificatio

Morpholinos for splice modification


Pathfinding in a large vertebrate axon tract: isotypic interactions guide retinotectal axons at multiple choice points
Andrew J. Pittman, Mei-Yee Law, Chi-Bin Chien


Navigating axons respond to environmental guidance signals, but can also follow axons that have gone before - pioneer axons. Pioneers have been studied extensively in simple systems, but the role of axon-axon interactions remains largely unexplored in large vertebrate axon tracts, where cohorts of identical axons could potentially use isotypic interactions to guide each other through multiple choice points. Furthermore, the relative importance of axon-axon interactions compared with axon-autonomous receptor function has not been assessed. Here, we test the role of axon-axon interactions in retinotectal development, by devising a technique to selectively remove or replace early-born retinal ganglion cells (RGCs). We find that early RGCs are both necessary and sufficient for later axons to exit the eye. Furthermore, introducing misrouted axons by transplantation reveals that guidance from eye to tectum relies heavily on interactions between axons, including both pioneer-follower and community effects. We conclude that axon-axon interactions and ligand-receptor signaling have co-equal roles, cooperating to ensure the fidelity of axon guidance in developing vertebrate tracts.


To generate a functional nervous system, widely separated groups of neurons must form appropriate connections. Each newborn neuron extends an axon tipped by a motile growth cone, which navigates through sequential `choice points' to eventually reach its target (Dickson, 2002; 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.



All zebrafish were of the Tü or TL strains. The transgenic lines and mutant alleles used were: Tg(isl2b:GFP)zc7, Tg(isl2b:mCherry-CAAX)zc23 or Tg(isl2b:mCherry-CAAX)zc25 (both of similar brightness), Tg(brn3c:gap43-GFP)s356t (Xiao et al., 2005), 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 1× 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 1.6× 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 (; 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).


To test the role of pioneer neurons, we devised a method to selectively remove early-born RGCs. These cells' differentiation starts ventroanteriorly at 27-28 hours postfertilization (hpf), spreading circumferentially to fill the central retina by ∼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.

Fig. 1.

ath5 MO blocks differentiation of early- but not late-born RGCs. (A) Normally, early RGCs differentiate in a wave across central retina (blue arrow); late RGCs are then added centrifugally (red arrows). (B,C) 6 dpf isl2b:GFP, lateral views, anterior towards the left. Insets schematize cell bodies, axons and optic nerve head (star). (B) Wild-type eye shows GFP+ RGCs throughout central retina; axons are obscured by cell bodies. (C) A high dose of ath5MO blocks differentiation of early RGCs, but late RGCs still form (arrows). Without central RGCs, peripheral axons are visible (arrowheads). (D) Dose-response curve showing timing of RGC formation with different doses of ath5MO. In wild type and with 3 ng control MO, GFP+ RGCs appear by 33 hpf. Increasing concentrations of ath5MO increasingly delay the appearance of the first RGCs. A, anterior; D, dorsal; P, posterior; V, ventral. Scale bar: 50 μm.

Fig. 2.

Early RGCs are necessary for axons to exit eye. (A) Diagram showing field of view for B,C. (B,C) 5 dpf isl2b:GFP, confocal z-projections; dorsal views, anterior upwards. Insets show cell bodies in eye. (B) Wild-type axons exit eye (arrowheads), project through the optic chiasm (broken lines) and to the optic tecta. (C) With high dose of ath5 MO, axons do not exit eyes (arrowheads) or innervate tecta (outlined). (D) Retinal exit in dose-response experiment of Fig. 1D, plotting percentage of embryos in which axons exit the eye against the time at which their first isl2b:gfp-positive RGCs were born. When RGCs are born by 42 hpf, axons usually exit the eye; when delayed after 42 hpf, axons rarely exit. Number of embryos is indicated at base of each bar. (E) 72 hpf isl2b:GFP, confocal z-projection; dorsal upwards. In a high-dose ath5 morphant, axons from peripheral RGCs remain trapped in the RGC layer without entering the optic nerve. To better appreciate 3D structure, see volume reconstruction in Movie 1 in the supplementary material. Arrowhead indicates the optic nerve head, stained by anti-Pax2 (not shown). Scale bars: 100 μm in C; 50 μm in E.

We then tested whether removing early-born RGCs affects the pathfinding of later axons (Fig. 2). In uninjected controls, the retinotectal projection is mature at 5 dpf, with both optic tecta strongly innervated (Fig. 2B; n=12/12). However, when central RGCs were removed by injecting 5 ng ath5MO, axons of late-born RGCs failed to exit from the eye into the brain (Fig. 2C; n=19/20). We hypothesized that this failure might be related to the time of first RGC differentiation. Therefore, we injected isl2b:GFP embryos with 2-5 ng ath5MO (Fig. 1D), plotting retinal exit at 5 dpf against the time of first RGC differentiation (Fig. 2D). Axons projected from eye to tectum in 100% of control larvae (uninjected, n=12; 3 ng control MO, n=12) and in 83% (45/54) of those morphants (MO-injected embryos) whose first RGCs were born by 42 hpf. By contrast, axons exited the eye in only 5% (1/20) of morphants whose first RGCs were born after 42 hpf. Instead, axons from peripheral RGCs grew within the RGC layer of the eye without entering the intraretinal region of the optic nerve (Fig. 2E, see Movie 1 and Fig. S1 in the supplementary material). A physical barrier is not likely the cause: ath5 morphants have normal retinal lamination, and the presumptive optic nerve can still be distinguished (see Fig. S1 in the supplementary material). To test for changes in the environment of the presumptive optic nerve, we stained for Pax2, which labels the surrounding primitive glia (Macdonald et al., 1997), 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.

Fig. 3.

ath5 MO does not appear to affect the molecular and cellular environment of the optic nerve head or the intraretinal optic nerve. Coronal sections through 48-49 hpf isl2b:GFP (A,B) and 36 hpf non-transgenic (C,D) eyes, from either uninjected embryos (A,C) or embryos injected with high dose ath5 TMO (B,D). (A,B) Pax2a antibody staining (magenta) labels presumptive glial cells which line the intraretinal region of the optic nerve (arrows) in both wild type (A) and ath5 morphants (B). RGCs and their axons (green) are present in wild type (A), but not in ath5 morphants (B). The trigeminal ganglion (arrowheads) is also labeled by isl2b:gfp and serves as a staining control. (C,D) In situ hybridization shows that netrin1a expression (arrows) surrounds the optic nerve and optic nerve head in wild type (C), and is unchanged in ath5 morphants (D). Scale bars: 50 μm.

Fig. 4.

Transplanted WT central RGCs rescue retinal exit in ath5 morphants. (A) Blastula transplants from isl2b:mCherry donors resupply ath5 morphant hosts, labeled with isl2b:gfp, with early RGCs. RhDx, rhodamine-dextran cell lineage marker. (B) Resupplied WT RGCs and axons (magenta) are sufficient to rescue host axons in morphants (green). Pigment cell autofluorescence is seen around eyes and at dorsal midline (magenta). Retinal axons project across chiasm (broken lines); donor axons terminate in central tectum (asterisk), while host axons terminate in peripheral tectum (arrowheads). Dorsal view, 5 dpf. (C) Lateral view of 5 dpf eye showing peripheral host axons (green) fasciculating with resupplied WT axons (magenta). (C′) Donor and (C″) host axons, reverse contrast. Scale bars: 100 μm in B; 50 μm in C.

In a second series of experiments, we tested whether, after retinal axons exit the eye, interactions between them might affect guidance to the tectum. To do this, we used cell transplants to mix retinal axons of different intrinsic pathfinding potential. In astray (ast) mutants, retinal axons make drastic pathfinding errors because they lack the Slit receptor Robo2 (Fricke et al., 2001). 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.

Fig. 5.

Axon-axon interactions strongly influence retinotectal pathfinding. (A) Transplants yield GFP-expressing donor RGCs in host eyes. (B-E) Dorsal views, 5 dpf, rostral upwards. Donor axons labeled with brn3c:GFP; pathfinding errors indicated by yellow stars. (B′-E′) Error quantitation. (B,B′) In wild-type> wild-type transplants, donor axons pathfind perfectly. (C,C′) By contrast, when transplanted into ast hosts, wild-type axons make significantly more errors. (D,D′) As expected, ast donor axons make many errors in ast hosts. (E,E′) However, when transplanted into wild-type hosts, ast axons make significantly fewer errors. Scale bar: 50 μm.

Fig. 6.

Pioneer axons influence pathfinding of follower axons. (A) Transplants resupply early RGCs to brn3c:GFP hosts injected with ath5 MO. (B-E) Dorsal views at 5 dpf, rostral upwards. Host axons terminate peripherally on the tectum; errors indicated by stars. (B′-E′) Error quantitation. (B,B′) In wild type>wild type;ath5 MO transplants, host follower axons pathfind normally. (C,C′) When pioneers are replaced with ast cells, wild type follower axons make significantly more mistakes. (D,D′) In ast>ast;ath5MO transplants, host follower axons make many errors. (E,E′) When pioneers are replaced with wild-type cells, follower ast axons make fewer errors but are not completely rescued. Scale bar: 50 μm.


We have shown that early-born RGCs are both necessary and sufficient for retinal axons to exit the eye. This is a somewhat surprising result in light of an elegant study in Xenopus (Holt, 1984), 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


We thank members of the Chien laboratory for discussions, Jude Rosenthal for technical assistance, Michael Bastiani and Richard Dorsky for comments on the manuscript, Tong Xiao and Herwig Baier for the brn3c:gfp line, Alexander Picker for anti-Pax2, and Hideo Otsuna for help with cell counts and 3D visualization. This work was supported by grants from the NIH/NEI and the University of Utah Research Foundation to C.-B.C.


  • * These authors contributed equally to this work

    • Accepted June 25, 2008.


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