Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

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Visual information is conveyed from the retina to higher brain regions by retinal ganglion cells (RGCs), the only output from the retina. RGCs project toward the ventral diencephalon, where fibers from both retinas converge to form the X-shaped optic chiasm. RGC divergence at the chiasm establishes a binocular projection, which is essential for information from one point of visual space to be observed by both retinas yet converge at the same loci in higher visual areas. Throughout the animal kingdom, the extent of RGC divergence closely correlates with the degree of binocular vision. Uncrossed RGC axons always arise from the temporal portion of the retina, whereas crossed RGC axons always arise from nasal retina. In humans, a highly binocular species, about 40% of all RGC axons project ipsilaterally, whereas this percentage drops to around 15% in ferrets. In most birds and fishes, uncrossed projections are absent because the lateral location of their eyes does not allow for any overlap in visual space.

Mice are a poor binocular species, with a very small population of uncrossed RGC fibers (ca. 3%–5%, depending on the strain of mice). These uncrossed axons arise from the peripheral ventrotemporal (VT) crescent. Despite the small number of uncrossed axons, the murine retina is an excellent model for developmental studies (Guillery et al., 1995). Over the past three decades, research on the mouse, in conjunction with work on fish, frog, and chick models, has uncovered molecules that pattern the retina and guide RGC axons through the visual pathway to their final destination. Moreover, in vitro and in vivo assays utilizing the murine visual system have been successfully combined with genetic approaches to study the molecular mechanisms guiding retinal divergence at the optic chiasm.

In this chapter, we examine the development of RGC projections from the retina through the optic tract. First, we review the development of ipsilaterally and contralaterally projecting RGCs during the early, middle, and late period of growth. Next we emphasize the cells, guidance cues, and regulatory genes that direct RGC projections throughout the visual pathway. We then discuss differences between mechanisms guiding uncrossed versus crossed projections by focusing on recent studies that have uncovered molecular mechanisms important for axon divergence at the chiasm.

Throughout, we highlight perturbations of molecular expression in the retina that alter the proportion of crossed to uncrossed fibers. These perturbations are of special interest in albino mutants, a unique case in which both the proportion of ipsilateral projections and the line of decussation (the border in the temporal retina demarcating the ipsilaterally projecting RGCs) are altered. The work reviewed in this chapter highlights the mouse visual system as an excellent model for understanding the development of retinal projections.

Development of retinal ganglion cell projections: Early, middle, and late stages

In the murine retina, RGCs differentiate shortly after optic fissure closure, around embryonic day 11 (E11), and continue to differentiate until about P0. RGC differentiation occurs in a central to peripheral gradient, such that the youngest RGCs are added in the peripheral retina. RGC projections can be divided into three phases, with each phase displaying different trajectories at the optic chiasm.

Early Phase The earliest-born RGCs are located in the dorsocentral (DC) retina and are referred to as “pioneer axons” because they are thought to help establish the projection pathways of later-born RGC axons. The DC fibers enter the ventral diencephalon at E12–E13, where they give rise to both crossed and uncrossed projections (Colello and Guillery, 1990; Marcus et al., 1995). The crossing fibers project through the radial glia palisade and follow the contour of a population of early-born chiasm neurons, which forms an inverted V-shaped array at the posterior boundary of the future X-shaped optic chiasm (figure 32.1A). During this early phase, uncrossed axons do not approach the midline glial palisade and instead project directly into the ipsilateral optic tract upon entering the ventral diencephalon. The pioneering ipsilateral projections from DC retina are transient, but whether the cells die or migrate to the VT retina, the site of mature ipsilateral projections, remains unknown.

Middle Phase RGC projections continue to enter the ventral diencephalon from E14.5 to E16.5, the peak phase

EphB1

Zic2

Nr-CAM

CD44/SSEA-1 Chiasm neurons

Ephrin-B2 radial glia

Figure 32.1 The three phases of RGC projections, and cell populations encountered by RGC axons. A, During the early phase of RGC axonogenesis, crossed projections (EphB1−) and transient uncrossed projections (EphB1+) arise from the dorsocentral retina. The uncrossed fibers do not interact with the midline radial glia, instead projecting directly into the ipsilateral optic tract, while the crossed projections follow the border of the early-born chiasm neurons. B, EphB1 and Zic2 are upregulated in the VT crescent during the middle phase, giving rise to the uncrossed RGC projections found in the adult. C, At later ages, RGC projections from VT retina cross the chiasm, concurrent with Nr-CAM upregulation in the VT crescent. D, Coronal section through optic disc immunostained for Pax2 highlights the optic disc astrocyte precursor (ODAPs) population surrounding RGC axons exiting the retina (Courtesy of T. Petros and C. Mason.) E, Interfascicular glia are clearly visible surrounding axon bundles in this transverse section through the optic nerve, as visualized with GLAST immunostaining (Courtesy of T. Petros.) F, RC2+ processes of midline radial glia extend from the base of the third ventricle to the pial surface of the ventral diencephalon, directly through the pathway of RGC projections (Courtesy of S. Williams.) White asterisks indicate location of RGC axons.

of RGC outgrowth. RGC axons arising from non-VT retina project through the optic chiasm into the contralateral optic tract. Unlike in the early phase, uncrossed RGC fibers arise from the peripheral VT crescent and enter the glial palisade near the midline (figure 32.1B). Time-lapse imaging of DiIfilled RGCs demonstrates that growth cones from VT retina

pause, often for hours, and undergo dynamic extension and retraction before making a U-turn back into the ipsilateral optic tract. Although growth cones from non-VT RGCs also pause before crossing, they do not display such extensive exploratory behavior (Godement et al., 1994; Mason and Wang, 1997).

Late Phase Crossed RGC projections arise from the expanding peripheral retina until birth (E19), and their projections follow earlier-born RGC axons into the contralateral optic tract. During this late phase (E17–P0) of RGC development, the vast majority of newly born VT RGCs project contralaterally rather than ipsilaterally (Dräger, 1985) (figure 32.1C). This temporal change in projection pattern mimics the late development of the cat retina (Reese et al., 1991), in which early-born temporal RGCs project ipsilaterally and later-born temporal axons project contralaterally, implying a conserved evolutionary mechanism.

Glia in the retinal ganglion cell axon projection pathway

Optic Disc Astrocyte Precursor Cells The majority of RGC projections exit the retina before a glial network develops. However, optic disc astrocyte precursor cells (ODAPs) are a specialized population of Pax2+ astrocyte precursor cells (APCs) that encircle RGC axons as they exit the retina (figure 32.1D). ODAPs give rise to astrocytes that migrate into the retina at later ages and populate the RGC fiber layer (Watanabe and Raff, 1988; Dakubo et al., 2003; Petros et al., 2006). Mice lacking ODAPs display hypoplasia and severe RGC projection errors in the retina (Dakubo et al., 2003), demonstrating the importance of these cells in guiding RGCs out of the retina.

Astrocyte Progenitors/Astrocytes in Optic Nerve

Upon entering the optic nerve, RGC axons fasciculate and form bundles as they project toward the ventral diencephalon. These bundles are ensheathed by interfascicular glia (Colello and Guillery, 1992), which are most likely APCs that give rise to the astrocytes that populate the mature optic nerve (figure 32.1E). The molecular mechanisms by which these interfascicular glia organize and guide RGC axons toward the optic chiasm are unknown. Around E18, specialized astrocytes at the optic nerve head form a tight meshwork that appears to act as a barrier to oligodendrocytes migrating toward the retina (similar to the lamina cribrosa in other species). Although these specialized glia become reactive in retinal diseases such as glaucoma (reviewed in Hernandez, 2000), their role in the development of RGC projections has not been addressed.

Midline Radial Glia The organization of RGC axon bundles ensheathed by interfascicular glia changes at the

optic nerve–chiasm junction, at which point interfascicular glia are no longer observed. Instead, radial glia cell bodies line the base of the third ventricle and extend dense processes from the ventricular zone to the pial surface of the ventral diencephalon on either side of the midline (reviewed in Mason and Sretavan, 1997; figure 32.1F). These radial glia cells express proteins common to immature radial glia such as RC2 (specific to mouse radial glia), brain lipid-binding protein (BLBP), and the glutamate transporter GLAST (Williams et al., 2003). DiI labeling reveals that newly arrived RGC growth cones dive toward the pial surface and extend along the endfeet of the radial glia (Colello and Coleman, 1997; Chung et al., 2008). As RGC axons enter this palisade of glial processes, they segregate into crossed and uncrossed components (Marcus et al., 1995). Crossing axons traverse the midline midway along the radial glial processes, whereas uncrossed axons make their turn above the pial endfeet (Chung et al., 2008). Axon outgrowth from VT but not DT explants is substantially decreased when grown on dissociated chiasm midline cells (Wang et al., 1995), making these radial glia cells of primary interest for studying molecular mechanisms of axon divergence.

Guidance cues and their role in retinal ganglion cell axon navigation through the visual pathway

In the past decade, the identification of guidance factors in numerous species and systems has led to characterization of their role in the mouse retinal axon pathway (reviewed in Williams et al., 2004; figure 32.2). In this section, the discussion is limited to cues that are important for general RGC growth through the chiasm but that do not influence divergence at the chiasm. As detailed in the next several sections, studies of these guidance factors have relied on analysis of mutant mice, leading to several characteristic phenotypes such as axon stalling, projection errors, and axon defasciculation (figure 32.3).

Exiting the Retina In the retina, RGC growth cones must correctly integrate many guidance cues to properly orient toward the optic disc, where they turn 90° and enter the optic nerve. Chondroitin sulfate proteoglycans expressed in a high peripheral-low central gradient are required for orienting RGC axons toward the optic disc and away from the peripheral retina (Brittis et al., 1992). Both the BMP-1B receptor and EphB2/B3 receptors are expressed in a high ventral-low dorsal gradient. It is interesting that RGC axons from ventral retina make projection errors at the optic disc in Bmpr1b−/− mice (Liu et al., 2003), whereas EphB2/B3−/− display guidance errors from dorsal retina (Birgbauer et al., 2000). Two members of the immunoglobulin gene family, L1 and NCAM, are highly expressed on RGC axons, and perturbation of these molecules causes RGC growth cones

Figure 32.2 Cues that guide RGC axons through the visual pathway. Top, RGC axons (blue) express many receptors for a number of cues in the retina that guide them toward the optic disc and out of the retina. Bottom, A variety of cues surrounding the optic nerve, chiasm, and tract help to funnel RGC axons properly through the ventral diencephalon toward their final targets, the LGN and SC. ODAPs, optic disc astrocyte precursor cells. See color plate 21.

to misproject or stall in the retina (Brittis et al., 1995). ODAPs express several molecules that potentially interact with RGC axons at the optic disc, such as netrin-1 (Deiner et al., 1997), EphA4 (Petros et al., 2006), and Slit2 (Erskine et al., 2000). Whereas netrin-1 is clearly required for RGC axons to properly exit the retina (Deiner et al., 1997), the role of EphA4, Slit2, and other unidentified guidance cues in ODAPs remains unknown. Recently it has been demonstrated that the repulsive action of Slit1/2 expressed by the lens is important for several aspects of intraretinal RGC projections via interaction with roundabout (Robo) receptors on RGC axons (Thompson et al., 2006b).

Projecting through the Optic Nerve Once the RGC axons exit the retina, less is known about cues that guide projections through the optic nerve. Sema5A is expressed by neuroepithelial cells surrounding the optic nerve, and blocking Sema5A signaling leads to RGC axons straying out

of the optic nerve (Oster et al., 2003; figure 32.3B). RGC axons project through the interfascicular glia/APCs that express several guidance cues, such as EphA4 (Petros et al., 2006) and netrin-1 (Deiner et al., 1997), but it is unclear how these cues support RGC axon extension through the optic nerve.

Shaping the Chiasm RGC axons are guided toward the optic chiasm by cues expressed by the ventral diencephalic cells. Netrin-1, expressed by cells caudal to the chiasm, is required for RGC axons to enter the optic chiasm at the proper angle (Deiner and Sretavan, 1999; figure 32.3C). Slit1 is expressed by cells anterior to the chiasm, channeling RGC

axons toward the chiasm midline (Plump et al., 2002). Slit2 is strongly expressed directly dorsal and anterior to the proximal optic chiasm and is inhibitory to RGC axons (Erskine et al., 2000). Although Slit1 and Slit2 single-knockout mice display relatively normal phenotypes, Slit1/2 double mutants display an ectopic chiasm just anterior to the normal chiasm (through the normally Slit2+ region) and a subset of RGC axons misproject just lateral to the optic nerve–chiasm junction (through the normally Slit1+ regions) (Plump et al., 2002; figure 32.3D). Thus, Slit1/2 act as repulsive cues funneling Robo+ RGC axons toward the chiasm midline and optic tracts. Of note, RGC axons in mice lacking Mena, an actin regulatory protein expressed in RGC growth cones,

defasciculate at the chiasm and form an ectopic posterior chiasm, similar to the ectopic anterior chiasm observed in Slit1/2−/− mice (Menzies et al., 2004; figure 32.3E); however, the role of Mena in this signaling pathway is not understood.

An enzyme involved in the addition of sulfates to heparan sulfate (HS) sugars (Hs2st) is expressed just posterior to the optic chiasm midline, and RGC projections in Hs2st−/− mice wander posterior to the chiasm midline (Pratt et al., 2006; figure 32.3F). Hs6st1 is expressed directly anterior and posterior to the optic chiasm, mimicking Slit1/2 expression patterns (Pratt et al., 2006), and Hs6st1−/− RGCs are less sensitive to Slit2 repulsion.

Optic Tract GAP-43 is an integral membrane bound protein that is highly expressed by developing neurons during axon growth. In GAP-43−/− mice, RGC axons appear to reach the chiasm normally, but they stall at the chiasm, and their projections into the optic tract are grossly perturbed (Strittmatter et al., 1995; figure 32.3G). Subsequent studies found that GAP-43 is required in RGC axons for mediating interactions with unknown guidance cues at the chiasm– optic tract junction, as GAP-43−/− axons display decreased outgrowth on lateral diencephalon cell membranes (Zhang et al., 2000). Whereas GAP-43 is required at the chiasm– optic tract junction, Slit1 and Slit2 appear to be required in more dorsal segments of the optic tract (see figure 32.3D). In Slit1−/− and Slit1/2−/− mice, aberrant branches of RGC axons project into the telencephalon at several points along the optic tract (Thompson et al., 2006a).

These findings highlight the fact that RGC growth cones must integrate a wide variety of attractive and repulsive cues to properly navigate from the retina through the chiasm and into the optic tracts (summarized in figure 32.2). This analysis does not include the primary termination sites of RGC projections, the lateral geniculate nucleus (LGN) and superior colliculus (SC), where many guidance cues play an important role in establishing retinotopic organization and eye-specific projections (reviewed in this volume; see chapters 28, 31, and 34.)

Axon-axon interactions, which likely play a major role in guiding axons through the visual pathway, should be mentioned even though the molecular bases of these interactions are not understood. Fasciculation and bundling are important for neighbor-neighbor interactions, while defasciculation may play a role in reorganizing axons, as occurs before and after traversing the midline (Chan and Chung, 1999). Axon-axon interactions are quite challenging to analyze both in vitro and in vivo, largely because the molecules most often implicated in axon-axon signaling are cell adhesion molecules (CAMs), a large family whose members can function through homophilic and heterophilic interactions. Since many cells express several members of this family, the

function of one member may be compensated for by other members, thus masking a functional role for certain CAMs in RGC guidance. For example, CAM family members L1, neurofascin, contactin, TAG-1 and NB-2 are all expressed in similar (though not identical) patterns in RGCs during development (Williams et al., 2006). Characterizing the molecular components involved in RGC axon-axon interactions for extension through the visual pathway and possibly for regulating divergence at the chiasm remains a major issue for understanding the development of RGC projections.

Molecules important for axon divergence at the chiasm: The uncrossed projection

Over the past several years, substantial progress has been made toward understanding the development of the uncrossed pathway. Initial identification of molecular cues that guide RGC axon divergence at the optic chiasm came from Xenopus. Ephrin-B expression was detected at the Xenopus optic chiasm when ipsilateral projections develop during metamorphosis, but it is absent at earlier stages when all RGCs project contralaterally. Ectopic expression of ephrinBs at the midline leads to a precocious ipsilateral projection (Nakagawa et al., 2000). These findings in Xenopus were extended to the developing visual projections in mice.

Ephrin-B1/ephrin-B2 A screen of Eph receptors and ephrins in the retina and ventral diencephalon identified two candidates for guiding ipsilateral projections (figure 32.4A). Ephrin-B2 expression in the chiasm region is first observed at E13 in radial glia cells at the chiasm midline, and expression remains high until E16, after which it is downregulated (Williams et al., 2003; figure 32.4A). This temporal expression pattern correlates precisely with the generation of mature ipsilateral projections (E13.5–E16.5). Perturbation of ephrin-B2 leads to a decrease in the number of ipsilateral projections, further supporting a role for ephrinB2 as an important midline repulsive cue for uncrossed RGC axons (Williams et al., 2003).

In the retina, EphB1 is the only Eph receptor specifically expressed in VT crescent RGCs from E13.5 to E16 (Williams et al., 2003; figure 32.4A). From E12.5 to E14, EphB1 is also expressed in the DC retina, the source of the transient early-born uncrossed RGC projections. However, DC projections do not approach ephrin-B2-expressing radial glia cells at the chiasm midline, and thus the function of EphB1 in these early-born RGCs remains unknown. EphB1−/− mice display a significant reduction in ipsilateral RGC projections (see figures 32.3H and 32.4A), and perturbations of EphB1 in vitro causes chiasm cells to be less repulsive to VT axons (Williams et al., 2003). Although EphB2 and EphB3 are

Figure 32.4 Cues that guide RGC projections from the VT crescent. A, Top, In situ hybridizations highlighting ephrin-B2 expression at the chiasm and EphB1 in DC and VT retina at E14, but restricted to VT retina by E15.5. Bottom left and center, Retinal explant cultures from VT retina display decreased axon outgrowth when grown on ephrin-B2 substrates compared to DT explants, an effect not observed with high concentrations of Fc. Bottom right, Whole-retina DiI fills reveal a significant decrease in uncrossed projections in EphB1−/− mice compared to wild-type or EphB1+/− mice. (Reprinted from Williams et al. [2003]. Ephrin-B2 and EphB1 mediate retinal axon divergence at the optic chiasm. Neuron 39:919–935, copyright 2003, with permission from Elsevier.) B, Left, Sections through E16.5 retina reveal Zic2 mRNA and protein specifically expressed in VT retina. Right, Whole-retina DiI fills demonstrate a significant decrease in uncrossed projections in Zic2kd/+ mice that is further

enhanced in Zic2kd/kd mice (Reprinted from Herrera et al. [2003]. Zic2 patterns binocular vision by specifying the uncrossed retinal projection. Cell 114: 545–557, copyright 2003, with permission from Elsevier.) C, Left, Nr-CAM is weakly expressed in VT during the peak-phase of RGC projections, but Nr-CAM is strongly upregulated in VT during the late phase. Top right, When semiintact preps are cultured in the presence of function-blocking Nr-CAM-Fc, a large increase in ipsilateral projections is observed (as revealed by whole-retina DiI fills). Bottom right, Rhodaminedextran retrograde backfills reveal a decrease in contralaterally projecting RGCs from VT at E18.5 and a corresponding increase in ipsilateral projections, which is not observed at earlier ages. (Reprinted from Williams et al. [2006]. A role for Nr-CAM in the patterning of binocular visual pathways. Neuron 50:535–547, copyright 2006, with permission from Elsevier.)

expressed in a high ventral-low dorsal gradient, EphB2/ EphB3−/− mice have normal ipsilateral projections. These findings demonstrate that EphB1 is critical for the formation of the uncrossed projection. A small population of uncrossed fibers remains in EphB1 mutants, indicating that other factors may also influence ipsilateral projections.

Zic2 The zinc finger transcription factor Zic2 was found to be necessary for directing ipsilateral projections at the chiasm (figure 32.4B). Like EphB1, Zic2 is expressed in VT retina primarily during the formation of uncrossed projections, from E13.5 to E16 (Herrera et al., 2003). Zic2 knockdown mice display significantly reduced ipsilateral projections (see figure 32.3H), while Zic2 overexpression is sufficient to render contralaterally projecting RGCs to be inhibited by chiasm cells (Herrera et al., 2003). Zic2 and EphB1 appear to colocalize in VT RGCs (Pak et al., 2004), and Zic2 overexpression upregulates EphB1 mRNA in RGCs (Lee et al., 2008). Thus, Zic2 appears to regulate EphB1 expression in VT retina, which in turn interacts with ephrin-B2 at the chiasm midline, resulting in repulsion and ipsilateral projections. Surprisingly, Zic2 is not expressed by the EphB1+ early-born DC RGCs that project ipsilaterally, highlighting a significant difference between the pioneering, transient DC RGCs growing during the early period and VT RGCs that form the permanent ipsilateral projection (figure 32.1A–C).

Molecules important for axon divergence at the chiasm: The crossed projection

The characterization of EphB1/ephrin-B2 as the principal repulsive receptor-ligand pair for uncrossed axons has elucidated the molecular mechanisms guiding crossed projections. Recent work examining late-born crossed VT RGCs has provided some insight into the molecular mechanisms of crossed projections, but identification of cues associated with crossing fibers from non-VT retina has proved more elusive.

Nr-CAM Late-born RGCs from VT project contralaterally. Concurrent with the development of these lateborn crossed projections, EphB1 and ephrin-B2, which are required for the uncrossed projection, are downregulated in VT and chiasm, respectively, by E17 (Williams et al., 2003). During the middle phase of RGC projections, Nr-CAM is highly expressed throughout the RGC layer except for the VT crescent, where its levels are reduced (Williams et al., 2006). By E17, Nr-CAM expression is upregulated in VT retina, coincident with the production of late-born contralateral VT projections (figure 32.4C). Nr-CAM −/− mice display an increase in the number of late-born ipsilateral projections arising from VT and a corresponding decrease

in contralateral projections (Williams et al., 2006; see figures 32.3I and 32.4C). However, contralateral projections from non-VT retina appear normal in Nr-CAM−/− mice, indicating that Nr-CAM is required for mediating the crossing of late-born VT RGCs but not non-VT crossed RGC axons, even though both populations express significant levels of Nr-CAM. It is worth noting that CAM family members neurofascin and TAG-1 are also upregulated in VT retina at E17.5, both of which may interact with Nr-CAM to guide late-born uncrossed projections (Williams et al., 2006).

Islet2 The LIM-homeodomain transcription factor Isl2 is expressed in RGCs in a high dorsal-low ventral gradient during the peak phase of RGC differentiation (Pak et al., 2004). Similar to Nr-CAM, Isl2 expression in the VT crescent is weak at E15.5 but greatly increases during the late phase of RGC projections at E17.5. In a lacZ reporter mouse, all Isl2-positive RGCs project contralaterally (Pak et al., 2004). Isl2−/− mice display a significant increase in ipsilateral projections specifically from VT retina (see figure 32.3I ), while no ectopic ipsilateral projections are seen arising from other parts of the retina, mimicking the phenotype of Nr-CAM −/− mice (Williams et al., 2006). Whether Isl2 regulates Nr-CAM expression remains to be analyzed.

Default Mechanism versus Crossing-Promoting Molecules in Non-VT Retina Late-born crossed RGC projections from VT retina are molecularly distinct from crossed RGCs outside the VT crescent. Both Nr-CAM and Isl2 are expressed throughout the RGC layer during the peak phase of RGC growth and become upregulated in VT retina during the late phase, but Nr-CAM and Isl2 mutants display RGC projection defects only from this late-born VT population. To date, there are few clues as to the molecular mechanisms that guide contralateral projections from nonVT retina.

Is crossing by non-VT retina a “default mechanism”— that is, do RGC axons that lack receptors to repulsive cues freely traverse the chiasm midline and enter the contralateral optic tract? Or are there as yet unidentified cues expressed at the chiasm that direct RGC axons to cross the midline? If so, would perturbation of these cues result in ectopic ipsilateral projections from non-VT retina? The failure to identify guidance cues that promote crossed projections in the mouse optic chiasm supports the idea that midline crossing occurs by default. However, the observations that RGC growth cones from non-VT retina pause and expand at the chiasm midline before crossing (Godement et al., 1994) and that RGC axons from all retinal regions are inhibited by chiasm cells in vitro (Wang et al., 1995) imply that the crossing mechanism is more complex. The CAMs L1, Nr-CAM, TAG-1, and neurofascin show strong expression in non-VT retina during the middle period of RGC

growth, suggesting that these L1 family members may have redundant or co-active roles in midline crossing. Additionally, CAMs are known to directly interact with other guidance families in CNS and PNS pathways (Bechara et al, 2000), thus complicating the issue even further.

Other guidance factors have been characterized in zebrafish, in which all RGCs normally cross the midline and project to the contralateral tectum. Sema3d is expressed anterior and posterior to the chiasm (Halloran et al., 1999), similar to Slit1/2 in mice. Both overexpression and knockdown of Sema3d produce ectopic ipsilateral RGC projections, causing RGC growth cones to pause and display exploratory behavior at the chiasm midline, a behavior not observed in wild-type embryos (Sakai and Halloran, 2006). In the zebrafish mutant belladonna (lhx2), the optic chiasm does not form and all RGC axons project to the ipsilateral tectum, making the transcription factor Lhx2 a possible upstream regulator of Sema3d (Karlstrom et al., 1996; Seth et al., 2006). Thus, at least in zebrafish, crossing does not occur simply from a lack of receptors for repulsive cues, but instead there are specific cues and regulatory genes that direct RGC axons across the midline into the contralateral optic tract.

The Belgian sheepdog has a recessive mutation that, similar to belladonna, results in an achiasmatic RGC projection (Williams et al., 1994). Furthermore, there are several reports of achiasmatic humans (Victor et al., 2000), providing evidence that crossing the chiasm may not be a default mechanism in primates. Are there similar mutations in mice that might shed light on molecular mechanisms that guide crossed RGC axons? The transcription factor Pax2 is expressed at the optic disc and throughout the optic stalk during development (Nornes et al., 1990). Among other defects, Pax2−/− mice are achiasmatic, with all RGCs projecting into the ipsilateral optic tract (Torres et al., 1996; see figure 32.3J). This finding sheds light on potential signaling components for midline crossing at the chiasm, but identifying molecular mechanisms that direct contralateral projections remains one of the most important and least understood questions regarding the developing retinal projections.

Regulatory genes in the retina and chiasm important for chiasm patterning

Recent studies have uncovered a number of regulatory genes important for patterning the retina, several of which also play an important role in mediating RGC axon divergence at the chiasm. Many of these genes are also expressed in the ventral diencephalon, and knockout models of these genes can display moderate to severe defects in the optic chiasm environment. Although it can be difficult to determine the cause of RGC misprojections (i.e., defects in RGCs, the chiasm, or both), these studies provide insight into the devel-

opment of RGC projections and the chiasm. They can also be useful in establishing a catalogue of phenotypes for ongoing genetic screens, which are becoming more common in the mouse nervous system.

VAX1/2 Two members of the ventral anterior homeobox family, Vax1 and Vax2, are expressed in the ventral retina during development. Vax1 expression is confined to cells in the optic disc and optic stalk during RGC differentiation (Mui et al., 2005), similar to the transcription factor Pax2 (Nornes et al., 1990) and EphA4 (Petros et al., 2006). RGC axons of Vax1−/− mice stall at the optic nerve–chiasm junction and fail to enter the ventral diencephalon (Bertuzzi et al., 1999; see figure 32.3K). Vax2 is expressed in a high ventrallow nasal gradient in the mouse retina, similar to EphB2 (Birgbauer et al., 2000), and Vax2−/− mice display defects in retinal patterning and targeting in the SC (Barbieri et al., 2002; Mui et al., 2002). However, the first group reported that ipsilateral RGC projections are eliminated in Vax2−/− mice (Barbieri et al., 2002), while the line of Vax2−/− mice used by the second group had no defects in ipsilateral projections at the chiasm (Mui et al., 2002). These differences in RGC divergence at the chiasm could result from the strain variations (mixed 129/C57Bl6 vs. C57Bl6, respectively) or from differences in Vax2 gene-targeting deletions.

Foxg1/Foxd1 The winged helix transcription factors Foxg1 and Foxd1 (also known as BF-1 and BF-2) have complementary expression patterns in the retina, with Foxd1 being restricted to the VT quadrant and Foxg1 expressed primarily in the nasal retina (Herrera et al., 2004; Pratt et al., 2004). Foxg1−/− mice display a large increase in ipsilateral projections, although the expression of EphB1 and the source of these ectopic ipsilateral projections remain unknown (Pratt et al., 2004; see figure 32.3L). Surprisingly, Foxd1−/− mice also have an abnormally large ipsilateral projection even though EphB1 and Zic2 expression in VT retina are absent in these mice (Herrera et al., 2004; see figure 32.3M). Unlike WT retina, ipsilateral projections in Foxd1−/− mice originate from the entire retina. These findings are complicated by the fact that Foxd1 is also expressed in the ventral diencephalon, and the optic chiasm region is grossly perturbed in Foxd1−/− mice (Herrera et al., 2004).

Brn-3b/3c The POU domain transcription factor Brn-3b is essential for the proper differentiation and survival of RGCs. Brn-3b−/− mice have substantially fewer RGCs than wild-type mice, and the surviving RGCs display guidance errors at many regions of the visual pathway (Erkman et al., 2000; see figure 32.3N). Of note, Brn-3b−/− mice display a significant increase in ipsilateral projections (Erkman et al., 2000; Wang et al., 2002), although it is not known from

which retinal region these ectopic ipsilateral projections arise. Surprisingly, the misrouted ipsilateral axons were not observed in Brn-3b−/−/3c−/− mice (Wang et al., 2002), while many of the other defects observed in Brn-3b−/− mice remain (see figure 32.3O). This finding implicates Brn-3c in promoting ipsilateral RGC projections, even through only 50% of RGCs express Brn-3c and Brn-3c−/− mice are not reported to have any retinal or RGC projection defects.

One confounding factor with the interpretation of such phenotypes is that a given gene may be expressed in both the retina and the optic chiasm (i.e., Foxd1). The presence of a gene in both RGCs and chiasm cells consequently makes it difficult to determine whether the RGC projection errors arise from perturbations in RGC axons, chiasm cells, or both. One way to address this issue is by performing in vitro mix-and-match cultures, whereby wild-type retinal explants are cultured with mutant chiasm cells, and vice versa (Herrera et al., 2004; Williams et al., 2006). Thus, chiasm phenotypes must be analyzed with regard to retinal perturbations and the topographic source of RGCs, as well as perturbations of chiasm cells and the ventral diencephalon.

between 6° and 14° into the temporal retina (Hoffmann et al., 2003).

The precise location of the line of decussation is essential for establishing the proper organization of visual space, and its location in different species correlates well with the degree of binocular vision. However, genes that define the line of decussation are not known. Moreover, it remains unclear how perturbations in melanin biosynthesis affect retinal gene expression and RGC divergence at the midline.

One interesting theory for establishing the line of decussation is that a signal expressed by nonretinal tissue at the temporal pole of the retina could control gene expression important for uncrossed projections. As binocular vision evolved and eyes rotated from the side to the front of the head, this signal could have altered the patterning of the retina to increase the number of uncrossed fibers and shift the line of decussation nasally (Lambot et al., 2005). To investigate this theory, it would be of interest to examine the expression of transcription factors and diffusible cues in tissue adjacent to VT or temporal retina in various species, especially albinos, that display different degrees of binocularity.

The albino

Albinism, also known as congenital hypopigmentary disease, is caused by a defect in the synthesis or packaging of melanin. The lack of pigment in the retinal pigment epithelium (RPE) leads to a variety of defects in the retina and visual projections, one of which is a decrease in the number of uncrossed RGCs (for a review, see Jeffrey and Erskine, 2005). Several studies utilizing various techniques have identified that the percentage of uncrossed RGC projections correlates with the degree of RPE pigmentation (LaVail et al., 1978; Rachel et al., 2002), with albino mice having about 40%–50% fewer ipsilateral projections (Rice et al., 1995). Of note, albino mice show a reduction in the number of Zic2-expressing cells, in agreement with a diminished ipsilateral projection.

Albino organisms provide a unique model because they are the only known mouse mutation in which there is both a decrease in the proportion of uncrossed axons and a shift in the line of decussation that divides uncrossed and crossed regions of retina (see figure 32.3P). All of the RGC projection errors at the chiasm described earlier result in an increase or decrease in the number of ipsilaterally projecting RGCs, but in none of these cases is there a shift in the dividing line between uncrossed and crossed RGCs. The uncrossed RGCs in the albino arise from the most peripheral VT retina, resulting in a temporal shift in the line of decussation. In contrast, embryonic monocular enucleations result in a decrease in uncrossed projections from the intact eye that is similar to the levels seen in albinism, but enucleations do not alter the line of decussation (Chan and Guillery, 1993). In albino humans, the line of decussation shifts

New approaches for studying mouse visual system development

As evidenced earlier, although mice are a poor binocular species with a very small percentage of uncrossed axons, they are a useful organism for studying the development of RGC projections. The ability to manipulate mouse genetics is a powerful tool that can be combined with in vitro assays for studying molecular mechanisms guiding RGC axon growth and guidance. Different regions of the retina can be cultured either adjacent to a zone (border assay) or on alternating stripes (stripe assay) of a substrate, or in the presence of chiasm cells (Williams et al., 2003; Petros et al., 2006; figure 32.5A). Time-lapse imaging provides a means for observing RGC growth cones in real time as they interact with cues in vitro that they may contact in their projection pathway in vivo (figure 32.5B).

It has been technically challenging to express genes of interest into RGCs in vivo. Recently, in utero electroporation has been successfully adapted for gene delivery into the embryonic retina (Garcia-Frigola et al., 2007; figure 32.5C). Expression remains high for weeks, and GFP is clearly visible throughout RGC axons. The ability to ectopically express genes in RGCs and follow their trajectory provides a powerful technique for studying murine RGC projections.

Conclusion

Over the past two decades, progress has been made toward understanding visual system development, with a focus on optic chiasm patterning. The murine system provides an