Eloísa Herrera 1, Lucia Brown 2, Jun Aruga 3, Rivka A. Rachel 14, Gül Dolen 1, Katsuhiko Mikoshiba 3, Stephen Brown 2, and Carol A. Mason *1
The decussation of retinal ganglion cell (RGC) projections at the optic chiasm is essential for the normal mapping of visual information in higher visual centers. This projection pattern ensures that each relay receives information from both eyes, a pattern critical for proper binocular vision.

The proportion of uncrossed to crossed retinal fibers varies according to the extent of binocular vision across species. The uncrossed component ranges from 40% of all RGCs in humans (Kandel et al., 2000) to less than 15% in ferrets (Cucchiaro, 1991; Thompson and Morgan, 1993) and about 3%–5% in mice, depending on the strain (Rice et al., 1995). RGCs with an uncrossed trajectory are located in the temporal retina, but in less binocular species, ganglion cells that project ipsilaterally reside in the ventrotemporal (VT) retina. In Xenopus, a subpopulation of ganglion cells in VT retina projects ipsilaterally only at metamorphosis (Hoskins and Grobstein, 1985; Mann and Holt, 2001). Finally, adult birds and fish do not have an uncrossed projection (O'Leary et al., 1983; Polyak, 1957) and thus lack binocular vision.

How the development of the binocular pathway is determined has been a longstanding enigma. Most recent work has focused on the localization of guidance factors that function in other species and settings, especially in axon navigation at the CNS midline, and their mechanism of action (Mason and Erskine, 2000). Among these are Slit2 (Erskine et al., 2000; Plump et al., 2002), Nr-CAM (Lustig et al., 2001), chondroitin sulfate proteoglycans (Chung et al., 2000), and ephrin-A's (Marcus et al., 2000), all of which have been localized to the specialized populations of neurons and glia at the chiasmatic midline and modulate different aspects of RGC axon guidance. The only molecule expressed at the chiasm midline that is directly involved in RGC divergence is ephrin-B2, which prevents midline crossing of VT retinal axons in both Xenopus and mouse (Nakagawa et al., 2000; Williams et al., 2003). Moreover, EphB1, a receptor for ephrin-B2, is expressed in a restricted pattern in VT retina during RGC divergence at the midline and is essential for the formation of the uncrossed retinal pathway (Williams et al., 2003). Despite these advances in elucidating mechanisms of RGC axon divergence, it remains unknown whether there might be regulatory genes within the retina that determine the binocular projection, perhaps by encoding expression of guidance factor receptors.

Combinatorial codes of transcription factor expression define cell subtypes in the spinal cord (Tsuchida et al., 1994; Lee and Jessell, 1999; Arber et al., 2000; Pierani et al., 2001; for review, see Jessell, 2000; Shirasaki and Pfaff, 2002). Several of these transcription factors direct projections of subsets of motoneurons to specific peripheral targets (Lin et al., 1998; Kania et al., 2000). Members of the LIM protein family would be good candidates for specifying RGC subtype and, in turn, axon guidance in the retina, because at least two of them, Islet1 (Thor et al., 1991) and Islet2 (Brown et al., 2000), are expressed in RGCs. Other transcription factors, such as SOHo (Deitcher et al., 1994), BF-1 and BF-2 (Hatini et al., 1994), Brn.3b (Xiang et al., 1995), and Vax2 (Schulte et al., 1999) are expressed along the nasotemporal or dorsoventral retinal axes. However, none of these proteins are expressed in patterns that reflect the retinal axon decussation.

The Zic genes, a family of zinc finger transcription factors homologous to the Drosophila pair rule gene odd-paired (opa) (Benedyk et al., 1994; Aruga et al., 1996), are apt candidates for specification of axon projection relative to the midline, since Zic genes are involved in the left-right asymmetry of the body plan (Brown et al., 1998; Kitaguchi et al., 2000; Purandare et al., 2002), and they are expressed in the developing eye. At embryonic day (E)9.5, Zic1, Zic2, and Zic3 are expressed in the optic vesicle and stalk. After E10.5, the expression of the three genes converges in the ciliary margin zone (CMZ) of the retina (Nagai et al., 1997) where retinal precursors reside.

Here, we show that in addition to Zic2 expression in proliferating retinal cells in the CMZ, Zic2, but not Zic1 or Zic3, is upregulated in differentiated ganglion cells in VT retina when the ipsilateral projection is formed (E14–E17). Genetically modified mice that express low levels of Zic2 (Nagai et al., 2000) display a reduced ipsilateral projection. Moreover, in gain-of-function experiments in vitro, Zic2 is sufficient to switch the outgrowth behavior of retinal axons from crossed to uncrossed patterns in response to inhibitory cues from chiasm cells. Finally, the proportion of Zic2-expressing cells correlates with the degree of binocular vision across species, implicating Zic2 function in patterning binocular vision throughout evolution. These results indicate that Zic2 contributes to RGC subtype identity by directing the retinal axon projection at the optic chiasm midline.

Results

Zic2 Is Expressed in the Ventrotemporal Retina during the Outgrowth of the Uncrossed Retinal Projection
To investigate the possibility that Zic family members are expressed differentially in RGCs with an uncrossed or crossed trajectory, we first examined Zic protein expression in retina at E16.5, since this stage is within the principal period of axon divergence near the optic chiasm. In addition, at this age, contra- and ipsilaterally projecting RGCs are located in distinct areas of the retina (Guillery et al., 1995). Extracts of peripheral VT retina, where the uncrossed population resides, and dorsotemporal retina (DT), where only crossed RGCs are located, were immunoblotted using an anti-pan-Zic antibody. 293T cells transfected with Zic1, Zic2, or Zic3 were used as controls. While VT retinal lysates revealed a strong Zic2 band, this protein was barely detectable in DT retinal lysates. Immunohistochemistry using specific antibodies against Zic1 or Zic2 (Brown et al., 2003) on E16.5 retinal whole mounts revealed that Zic2, but not Zic1, is expressed specifically in VT retina. In situ hybridization and immunohistochemistry on retinal sections confirmed that Zic2 mRNA and protein are selectively localized in neural retina in the peripheral VT segment (Figures 1B and 1C) . Moreover, RGCs are the only retinal cells that express Islet at this age (Galli-Resta et al., 1997), and double immunostaining of Zic2 and of Islet1/2 demonstrated that Zic2 is located in the ganglion cell layer (Figure 1C). Thus, Zic2, but not other Zic family members, is expressed in RGCs at E16.5 in the peripheral VT neural retina, the source of the uncrossed retinal projection.

 
Figure 1. Zic1, Zic2, and Zic3 Expression in Developing Retina

(A) Peripheral ventrotemporal (VT) and dorsotemporal (DT) lysates from E16.5 retinae, immunoblotted with α-pan-Zic. 293-Zic1, -Zic2, and -Zic3 transformed cell lines are shown as controls. A band corresponding to Zic2 is detected in the VT retinal lysate but is barely detectable in the DT lysate. The weak bands observed in VT and DT lysates at the Zic1 and Zic3 molecular weight level are probably due to Zic1 expression in the ciliary margin (CMZ) at this age.

(B) E16.5 retinal whole mount stained with α-Zic1 (a and b) or α-Zic2 (c). (b) Higher magnification of (a). (1–5) Higher magnification of each retinal segment in (c). Note that only the VT segment shows Zic2+ cells. Abbreviations: N, nasal; D, dorsal; T, temporal; V, ventral; NR, neural retina; and CMZ, ciliary margin. Scale bar: 500 μm.

(C) Horizontal sections of E16.5 retina. (a) Zic2 mRNA detected in ventral retina by in situ hybridization (blue). (b) Zic2 in ventral retina (green, white arrows) detected by α-Zic2. (c) RGC layer (red) visualized with α-Islet1/2 in the same section as (b). Scale bar: 100 μm.

(D) Zic2 is expressed in postmitotic cells in VT retina. (a–d) Confocal sections of the VT region labeled with α-Islet1/2 (a, red), α-Zic2 (b, green) or α-BrdU (c, blue). (d) merged image; Zic2+ cells in the neural retina are also Islet1/2+ (yellow), but rarely BrdU+ (turquoise), as in the CMZ. Abbreviations: NR, neural retina; CMZ, ciliary margin zone. Scale bar: 100 μm.
 

In mouse, RGCs are generated from E11.5 to birth. The first RGCs to be born and project axons both ipsi- and contralaterally derive from dorsocentral retina, but this uncrossed RGC population is thought to be transient. The permanent ipsilaterally projecting RGCs arise from VT retina, and these are generated from E14.5–E17.5. In contrast, contralaterally projecting cells differentiate at E11.5 and continue proliferating until the end of embryogenesis (Drager, 1985; Colello and Guillery, 1990). To determine whether the timing of Zic2 expression relates to generation and formation of the ipsilateral projection, E13.5–E18.5 retinal whole mounts were immunostained with anti-Zic2 antibodies. At E13.5, no Zic2-positive cells were found in any retinal quadrant except for weak staining in the CMZ around the entire retina (Figure 2 Ba) (Nagai et al., 1997). Strong expression of Zic2 in the neural retina was first detected in the VT segment at E14.5 (Figure 2Bb), when axons from the VT retina begin to grow ipsilaterally to form the permanent uncrossed projection. For the next 2 days, the number of Zic2-positive cells increases as additional ipsilaterally-projecting RGCs in VT retina grow into the optic stalk. From E17.5–E18.5, when the generation of ipsilaterally projecting RGCs terminates, Zic2 expression wanes (Figure 2Bd). Zic2 is thus expressed in RGCs in a highly restricted spatiotemporal pattern, precisely at the time the uncrossed RGC projection from VT neural retina is established

 
Figure 2. Zic2 Is Expressed from E14.5–E18.5

(A) Schematic representation of the VT segment of the retina, indicating the central to peripheral location of Zic2 over time. ON, optic nerve.

(B) In (a)–(d), VT retinae from E13.5-E18.5 mouse embryos, stained with α-Zic2. (a) At E13.5, no Zic2+ cells are detected in the VT neural retina. Note weak staining at the ciliary margin (CMZ) at this age, detected only until E14.5–15.5. (b) At E14.5 Zic2 is upregulated in VT neural retina. (c) A peak in the number of Zic2+ cells in VT retina is observed at E16.5, but by E18.5, few Zic2-expressing cells are seen (d). Scale bar: 100 μm
 

Zic2 Expression in Ventrotemporal Retina Is Postmitotic
Neuronal differentiation is controlled by transcription factors acting either during neuronal proliferation to specify neuronal fate (Cepko et al., 1996) or in postmitotic neurons to specify neuronal subtype (for review, see Shirasaki and Pfaff, 2002). Zic2-expressing RGCs are always located peripherally at a gradually increasing distance from central retina, in a pattern mirroring RGC genesis (Figure 2A). To investigate whether Zic2 is expressed in neural VT retina in differentiated RGCs or in proliferating cells, we labeled cycling cells with BrdU and then colabeled retinal whole mounts with antibodies against Islet 1/2 and Zic2. Islet1 and Islet2 are expressed by postmitotic RGCs as they leave the cell cycle (Rachel et al., 2002). At all ages studied (E14.5, E16.5, and E18.5), Zic2-positive cells within the RGC layer in VT retina were Islet1/2 positive but rarely BrdU labeled (Figure 1D). Thus, Zic2 is expressed in postmitotic RGCs in VT neural retina, supporting a role for Zic2 in the specification of the uncrossed RGC subtype.

Zic2 Is Expressed Exclusively in Uncrossed Retinal Ganglion Cells
To investigate whether Zic2 is expressed exclusively in ganglion cells that project ipsilaterally, RGCs were retrogradely labeled with rhodamine dextran from one optic tract in E16.5–E18.5 embryos, and retinal whole mounts were immunostained with anti-Zic2. In the retina ipsilateral to the labeled optic tract, retrogradely labeled cells were located in the VT retinal segment (Figure 3A) but positioned more centrally than Zic2-positive cells. This pattern indicates that the ganglion cells labeled from the optic tract are more mature than Zic2-expressing ganglion cells, since the wave of RGC generation progresses from central to peripheral retina. However, even though the majority of retrogradely labeled and Zic2-positive cells are not in exact register, at E16.5, a region exists where both populations overlap (Figure 3A); these double-labeled cells represent approximately 5% of all retrogradely labeled RGCs.

 
Figure 3. Zic2-Positive Cells and RGCs Retrogradely Labeled from the Ipsilateral Optic Tract Are Located in the Same Zone but Do Not Always Colabel

(A and B) Retrogradely labeled retinae, stained with α-Zic2. (A) Retina ipsilateral to the labeled optic tract. (B) Retina contralateral to the labeled optic tract. (a) Diagram representing cells labeled retrogradely from the optic tract (OT) (red). (b) View of half of a retinal whole mount showing retrogradely labeled cells (red) and Zic2+ cells (green). (c) Higher magnification of (b); note that only in the ipsilateral, but not in the contralateral, retina Zic2+ cells (green) are intermixed with dextran+ cells (red), and occasionally some cells are double labeled (yellow). Abbreviations: NR, neural retina; CMZ, ciliary margin zone. Scale bar: 100 μm.

(C) Schema summarizing the spatial aspects of Zic2 expression (green dots) with respect to zones occupied by Islet1/2+ RGCs (blue) and RGCs retrogradely labeled from the optic tract (red dots). In both eyes, Islet1/2 is expressed by RGCs in the entire retina, whereas Zic2 is observed in the peripheral VT retina. In the eye ipsilateral to the dextran label, RGCs are labeled only in VT retina and are located more centrally than Zic2+ cells. Yellow dots mark double-labeled cells. In the contralateral retina, dextran+ cells are found in the entire retina except in the region in VT retina where uncrossed RGCs from this eye should be located. Instead, there is a gap between dextran-labeled and Zic2+ populations. Cells located in the gap are Islet1/2+, indicating that they are RGCs.
 

In the retina contralateral to the labeled optic tract, dextran-labeled cells were found throughout the retina with the exception of the peripheral VT retina (Figure 3B), where cells double-labeled with Zic2 and dextran were never observed (Figure 3B). This Zic2- and dextran-label-free gap in the contralateral retina presumably contains the RGCs that project ipsilaterally from this eye. By E17.5, however, the gap between contralaterally projecting dextran-labeled cells and Zic2-positive RGCs is almost imperceptible (data not shown). This latter finding is consistent with previous data showing that up to E17.5–18.5, crossed projections originate from RGCs over the entire retina except for the VT region; RGCs born after this time are only contralaterally projecting and intermingle with already differentiated ipsilaterally projecting RGCs in VT retina (Guillery et al., 1995).

These results on the time course of Zic2 expression coinciding with the generation of the ipsilateral projection, together with the colocalization of Zic2 and retrograde labeling in the ipsilateral, but not contralateral retina, argue that Zic2 is exclusively expressed in RGCs that project ipsilaterally.

Zic2 Expression Is Downregulated As RGCs Extend toward Chiasmatic Midline
The retrograde labeling experiments described above show that at E17.5, only a small proportion of cells within VT retina that were labeled from the ipsilateral optic tract express Zic2 (<5% of all ipsilateral cells, Figure 3A). One explanation for this observation is that Zic2 is initially expressed in RGCs that will ultimately project ipsilaterally but is downregulated once axons reach the optic chiasm. Alternatively, it may not have been possible to retrogradely label many of the Zic2-positive cells that project ipsilaterally because their axons have not yet reached the optic tract. To distinguish between these possibilities, we applied dextran to the optic tract at E15.5, E16.5, and E17.5 and compared the total number of retrogradely labeled ganglion cells with the number of Zic2-positive cells in the retina ipsilateral to the injected optic tract. At E15.5, the Zic2-expressing population was larger than the number of dextran-labeled cells (578 ± 96 Zic2-positive cells/retina compared to 285 ± 24 dextran-positive cells/retina), and both numbers nearly doubled by E16.5. However, by E17.5, the number of cells dextran-labeled cells exceeded the number of Zic2-positive cells (668 ± 86 Zic2-positive cells/retina and 1284 ± 88 dextran-positive cells/retina) (Figure 4A) . These data suggest that at E17.5, when many more uncrossed fibers have reached the optic tract compared to E15.5, Zic2 expression is downregulated in the retina.

 
Figure 4. Zic2 Is Downregulated before Axons Enter the Optic Tract

(A) Quantification of Zic2+ cells (green line) and RGCs retrogradely labeled from the ipsilateral optic tract (red line) at E15.5, E16.5, and E17.5 in retinal whole mounts. At earlier ages, more Zic2+ cells are detected relative to the number of dextran-labeled uncrossed cells. In contrast, at E17.5, the number of ipsilateral cells labeled with dextran exceeds the number of Zic2+ cells (n = 5 retinal whole mounts for each age).

(B) In (a)–(d), confocal sections of VT retinal whole mounts retrogradely labeled with rhodamine dextran at E17.5 (red) and stained with α-Zic2 (green). The application point of dextran is indicated above each picture. Red arrow indicates the eye used to count RGCs. Scale bar: 100 μm.

(C) Quantification of double-labeled RGCs (yellow cells in Figure 4B) after retrograde labeling and α-Zic2 staining. The number of double-labeled cells was counted in confocal sections. As the site of dextran application became more distal to the retina, fewer RGCs were double-labeled. Number above bar indicates number of sections from five different retinae. Abbreviations: OD, optic disc; ON, optic nerve; Ch, optic chiasm; and OT, optic tract.

(D) Schema summarizing the spatiotemporal features of Zic2 expression in the developing neural retina. Zic2 is expressed postmitotically, through RGC extension to and within the chiasm and then is downregulated in the optic tract.
 

To determine where along the route Zic2 expression might be downregulated, RGC axons were retrogradely labeled via four points along the retinal axon trajectory: (1) the optic tract, after RGC axons exit the chiasm; (2) the optic chiasm; (3) the optic nerve, just before the axons reach the chiasm; and (4) the optic disc, as axons are leaving the retina. Retinal whole mounts were incubated with anti-Zic2 and the number of double-stained cells counted in the eye ipsilateral to the dextran application. It should be noted that dextran application into the optic chiasm, nerve, or disc labels RGCs that project both ipsi- and contralaterally. However, based on the Zic2/dextran-free gap observed in the retina contralateral to optic tract labeling (see above), we assumed that any double-labeled cells in this zone must be RGCs that project ipsilaterally.

As described above, Zic2 is expressed only in a few cells retrogradely labeled from the ipsilateral optic tract (1.75 ± 1.30 double-labeled cells/section or 5% of all retrogradely labeled ipsilateral cells) (Figure 4Ba). Retrograde labeling from the chiasm yielded a greater number of dextran-positive cells that expressed Zic2 compared to labeling from the optic tract (5.3 ± 1.7 double-labeled cells/section, representing approximately 15% of all retrogradely labeled cells that projected ipsilaterally) (Figures 4Bb and 4C). Application of dextran to the optic nerve resulted in yet more cells double labeled for Zic2 and dextran (11.4 ± 2.1 double-labeled cells/section or 32% of all retrogradely labeled RGCs projecting ipsilaterally) (Figures 4Bc and 4C). When axons were labeled from the optic disc, the number increased to 19.0 ± 3.02 double-labeled cells/section (54% of all retrogradely labeled ipsilaterally projecting RGCs) (Figures 4Bd and 4C). Thus, the closer to the eye the label was applied, the more dextran-labeled cells were observed that expressed Zic2. These findings favor the hypothesis that Zic2 is expressed in RGCs that will project ipsilaterally during the early axonogenesis but is downregulated as axons navigate through the optic nerve and chiasm and is reduced to very low levels when RGCs reach the optic tract.

Zic2 Expression Reflects Different Degrees of Binocularity across Species
We hypothesized that if Zic2 specifies the ipsilateral projection, then the proportion of ganglion cells expressing Zic2 would reflect the relative size of the uncrossed projection in organisms having a larger or smaller ipsilateral retinal projection. We used four models to test this hypothesis: (1) albino mouse, where the proportion of uncrossed axons is reduced compared to the uncrossed component in pigmented mouse; (2) ferret, in which the uncrossed RGC zone is expanded compared to that in mouse; (3) Xenopus, in which an ipsilateral projection develops only after metamorphosis (about stage 54); and (4) chick, which lacks a permanent ipsilateral projection.

In albino mice, fewer Zic2-positive cells were observed compared to pigmented retinae at E14.5 (125 ± 38 cells/retina in albino and 207 ± 32 cells/retina in pigmented littermates) and at E16.5 (700 ± 66 cells/retina in albino and 1108 ± 77 cells/retina in pigmented) (Figure 5A) . This decrease in the number of Zic2-positive cells in albino retina (a reduction of about 37%) closely parallels the reduction in uncrossed RGCs compared to pigmented adult retinae (Rice et al., 1995).

 
Figure 5. The Number of Cells Expressing Zic2 in Different Species Reflects the Degree of Binocular Vision

(A) Albino mouse. Retinal whole mounts from littermates of E16.5 pigmented (a) and albino mouse embryos (b) stained with α-Zic2. Scale bar: 100 μm. (c) Quantification of Zic2+ cells in pigmented (black columns) versus albino retinae (gray columns) at E14.5 and E16.5. Number above bars indicates number of retinal whole mounts. *p < 0.05 compared to pigmented retinae (Student's unpaired t-test).

(B) Ferret. Retinal whole mounts from E27.5 (a) and E34.5 (b) embryos labeled with α-Zic2. Dashed white lines delineate the area in which Zic2 is expressed. (c) Retinal whole mount from an E34.5 embryo retrogradely labeled with DiI from the optic tract. Dashed white lines delineate retrogradely labeled cell location. (d–g) High magnification of selected areas in (a), (b), and (c), respectively. Scale bars: (d), 100 μm; (e and f), 500 μm.

(C) Xenopus. In situ hybridization of frontal sections using Zic2-specific probes in premetamorphic (a), metamorphic (stage 54) (b), and postmetamorphic Xenopus (c). Black arrows, Zic2 mRNA in the ciliary margin; white arrows, Zic2 mRNA in the neural retina. Abbreviations: D, dorsal; V, ventral; T, temporal; and N, nasal. Scale bar: 100 μm.