Specific Expression of DSCAM in the Developing Xenopus Optic Nerve, Chiasm, and Tectum
Previous work from our laboratory showed that DSCAM immunoreactivity localizes to the plasma membrane surface of both RGCs within the retina and neurons in the optic tectum in Xenopus tadpoles at stage 45 (27). Without permeabilization, punctate DSCAM immunoreactivity was localized to the tectal neuropil where retinotectal axons and dendrites establish functional synaptic connections at this stage. These observations led us to further characterize DSCAM expression patterns across the Xenopus visual system as a means to inform us about its roles in the structural development of retinotectal circuits. For these experiments, we permeabilized tissues and co-immunostained sections of stage 45 to 46 tadpoles with antibodies to DSCAM (as in our previous study (27)) together with the anti-neurofilament protein antibody (Mab 3A10) that has been shown to label a subset of retinal axons (28). As observed on coronal tissue sections, DSCAM was highly expressed at the ventral region of the optic nerve (Fig 1a, b). The mean fluorescence intensity of both DSCAM and 3A10 immunoreactivity were measured across the ventral-dorsal axis of the optic nerve bundle (measurements started at the ventral side of the optic nerve then continued to the dorsal side as marked by the white dotted line on Fig 1b). DSCAM immunofluorescent signal progressively decreased as measurements were obtained along the dorsal regions of the optic nerve. We plotted the mean fluorescence intensity on a graph with the x-axis representing regions along the ventral to dorsal portions of the optic nerve (Fig 1d). This analysis quantitatively confirmed a high-ventral to low-dorsal graded pattern of DSCAM expression. In contrast, 3A10 labeled axons were strongly localized to the dorsal region of the optic nerve (border noted by white dotted line on Fig 1b) with lower intensity signal along the ventral side. 3A10 immunofluorescence exhibited a low-ventral to high-dorsal distribution (Fig 1d) in what appeared to be an inverse of the high-ventral to low-dorsal DSCAM immunoreactivity pattern. Analysis of retinal axons as they crossed the midline at the optic chiasm (white arrowhead, Fig 1c) showed that DSCAM immunoreactivity strongly localized at the ventral base of the chiasm. Here again, the intensity of DSCAM immunostaining was gradually decreased at more dorsal areas of the optic chiasm (white dotted line, Fig 1c, e). 3A10 immunoreactivity in axonal fibers continued to show a low-ventral to high-dorsal fluorescence intensity along the optic chiasm (Fig 1c, e).
Having encountered differential DSCAM expression along the ventrodorsal axis of the optic nerve and chiasm, we further characterized the distribution of DSCAM along the posteroanterior axis. In horizontal tissue sections, we found higher DSCAM immunofluorescence intensity specifically at the posterior region of the optic nerve bundle (Fig 2a) and optic chiasm (white arrowhead, Fig 2b). DSCAM immunofluorescence intensity was lower at the anterior portions of the optic nerve and chiasm compared to the posterior side, indicating a high-posterior to low-anterior pattern of DSCAM expression (Fig 2 c, d). Immunostaining with the 3A10 antibody showed an inverse pattern, where the fluorescence distribution of 3A10 staining appeared lower posterior and higher anterior at both the optic nerve and chiasm (Fig 2 a-d). It is important to note that we did observe DSCAM expression along a number of fibers stained with 3A10 (white arrows, Fig 2a), confirming that the DSCAM immunostaining identified RGC axon fibers. These results suggest that a subpopulation of RGC axon fibers rely on DSCAM as a potential mechanism to navigate the optic nerve pathway and cross the optic chiasm.
To further determine if axonal arbors terminating and branching in the optic tectum express DSCAM in a pattern similar to the high-ventral to low-dorsal pattern found along the optic nerve and chiasm, we performed immunostaining of whole brain cleared tissues to preserve the structural layout of axonal tracts and arbors innervating the tectum. Compared to brain sectioning, brain clearing is a powerful technique that permits obtaining a novel three-dimensional perspective of any potential gradient pattern of DSCAM expression within the intact tectum (24). Cleared tissue samples of stage 45 to 46 tadpoles were immunostained with DSCAM and 3A10 antibodies (Fig 3a). Our results revealed that the optic nerve (solid white and yellow arrowhead, Fig 3a), as well as sensory and motor cranial nerves throughout the tadpole head were stained by the 3A10 antibody (Fig 3a). Even at low magnification, we were able to observe DSCAM immunoreactivity along the optic nerve throughout its full extent up to the optic chiasm (solid white and yellow arrowhead, Fig 3a, b). At the optic tectal neuropil, strongest DSCAM immunoreactivity coincided with axon terminals labeled with the 3A10 antibody (empty white and yellow arrowheads at the tectum, Fig 3a). When we examined individual horizontal z-stacks at the level of the neuropil, the fluorescence intensity of DSCAM immunoreactivity was higher along axon terminals as revealed by the 3A10 co-immunostaining (solid white and yellow arrowheads, Fig 3b) and lower at fasciculated axon bundle tracts as they enter the neuropil (empty white and yellow arrowheads, Fig 3b). To better outline DSCAM expression patterns, whole intact brain tissues were dissected, cleared, and immunostained with the DSCAM and 3A10 antibodies. When viewing the orthogonal planes of the confocal images of cleared brain tissues, we found that DSCAM immunoreactivity colocalized with the 3A10 retinal axon marker both at the x- and y-orthogonal planes (solid white and yellow arrows, Fig 3c). The mean average fluorescent intensity of DSCAM expression along axon terminals were more substantially elevated compared to the DSCAM signal along axon fiber tracts (Fig 3d).
It is important to note that DSCAM expression along the optic tectum is distinct compared to other brain regions of the Xenopus CNS. Noticeably, strong uniform DSCAM expression was found in the olfactory bulb and telencephalic regions of the tadpole brain (white and yellow arrowhead at OB and TEL, Fig 4a, b) where spiny neurons are located (29). A prominent pattern of DSCAM immunoreactivity was detected along axonal tracts at the lateral regions of the hindbrain (white and yellow arrowhead at the HB, Fig 4a, d). We quantitatively measured the mean fluorescent intensity of DSCAM of two intact tadpole heads at various regions of the Xenopus brain. DSCAM immunoreactivity at the olfactory bulb (Fig 4a, b, e) was higher in intensity than in the telencephalic brain region, the hindbrain, and the optic tectum. Similar to the patterns observed in dissected brain tissues (Fig 3c), the intensity of DSCAM immunostaining was higher in RGC axons at their axon terminals and lower at axonal tracts entering the neuropil (Fig 4c, e). These results suggest that DSCAM is differentially expressed or transported throughout the CNS.
To determine whether the patterns of DSCAM expression in the optic nerve, chiasma and in the optic tectum correspond with differential DSCAM expression within the retina, we analyzed retinas of stage 45 tadpoles in cleared intact tissues and cryostat sections immunostained with DSCAM and the 3A10 antibodies (Fig 5a, b). As previously shown, DSCAM immunoreactivity was observed in the ganglion cell layer (GCL), inner plexiform layer (IPL) and inner nuclear layer (INL) of the Xenopus retina (27), with punctate DSCAM expression found around cell bodies within the GCL (Fig 5b). The majority of cell bodies immunostained with the 3A10 antibody localized to the GCL, adjacent to the IPL. However, as observed both in coronal sections and in cleared intact eyes, not all RGCs were immunopositive for 3A10 (Fig 5 a, c) indicating that only a subset of RGCs express neurofilament-associated proteins recognized by the 3A10 antibody. Analysis of coronal sections of retinas co-stained with DSCAM and 3A10 antibodies showed that some axon fibers exiting the eye along the optic fiber layer (Fig 5d) and the optic nerve head were immunopositive for both DSCAM and 3A10 (Fig 5e; empty arrowheads), although a number of 3A10 positive fibers did not express DSCAM (white arrowheads; Figs 5 d, e). These observations reveal a differential pattern of expression of DSCAM by RGC axons as they exit the eye. Moreover, different subsets of RGCs, including those that differentially express DSCAM and neurofilament-associated proteins recognized by the 3A10 antibody, appear to organize in distinct topographic order as they navigate along their pathway to their target in the optic tectum.
Dorsoventral Axon Sorting in the Xenopus Retinotectal System and DSCAM Effects on Topographic Segregation at the Optic Tectum
A graded distribution of molecular cues has largely been implicated in topographic mapping. Based on its differential distribution, it is likely that DSCAM collaborates with other guidance and cell adhesion molecules in the topographic organization of axon retinal fibers at multiple points along their path (30). Indeed, analysis of a mouse model of Down syndrome showed that DSCAM regulates eye-specific segregation of retinogeniculate projections at the target, in the dorsal lateral geniculate nucleus (20). Thus, to explore whether DSCAM is directly involved in retinotopic organization in the Xenopus optic tectum, we first characterized the projection and ordering of ventral and dorsal retinal fibers as they travel from the eye through the chiasm and into the brain (as depicted schematically in Fig 6a). A scrambled control fluorescein-tagged MO (to serve as a green fluorescent marker) and a control lissamine-tagged MO (red fluorescent marker) were electroporated separately to label dorsal and ventral RGCs, respectively (Fig 6b, c). Our results show that ventral RGCs project axon fibers that are positioned along the ventral portion of the optic nerve, while dorsal RGCs send axon fibers along the dorsal region of the optic nerve (Fig 6c, d, e). As axons of both ventral and dorsal RGCs enter and cross the chiasm and turn contralaterally into the tectum, we observed a shifting of fiber arrangement, with lissamine MO-labeled axon fibers that were originally positioned on the ventral side of the optic nerve intermixing and positioning more dorsally in the optic chiasm (Fig 6d, f). This inverted projection was also observed for the fluorescein MO-labeled axon fibers that originate in the dorsal portion of the retina, shifting more ventrally (Fig 6f). A complete inverted arrangement was observed for axons as they innervate the tectum, with ventral RGC axons entering the tectum through the dorsal branch and dorsal RGC axons projecting ventrally within the tectum (Fig 6g) in agreement with previous studies (31, 32). Based on these results, our immunohistochemical data indicates that specific DSCAM expression along the ventral portion of the optic nerve would coincide with axon fibers traveling on the ventral side of the optic nerve pathway prior to crossing at the tectum (Fig 1a and Fig 3b).
Analysis of axon terminals along the lateral-medial axis (as depicted schematically in Fig 7a), showed that ventral RGC axons (labeled with lissamine-tagged MO) innervate the tectum laterally, while dorsal RGC axons (labeled with Alexa 488 dextran) travel more medially, as shown for other species (9, 33). Indeed, ventral and dorsal RGC axons from tadpoles injected at stage 46 and imaged 48 hrs later showed correct topographic mapping but with a consistent degree of arbor overlap (average 20 μm) as shown in Fig 7b. When retinal neurons were labeled few days later, at stage 47, and imaged 48 hrs after, medial arbors were visibly separated from lateral arbors (data not shown). This separation between lateral and medial arbors in the Xenopus tadpole is consistent with observations in zebrafish larvae at 5 days postfertilization, when the optic tectum is first fully innervated (9). Thus these in vivo imaging studies confirm that in Xenopus, dorsal RGC axons projecting through the lateral branch initially overlap with ventral RGC axons traveling through the medial branch; then, as the tectum expands and arbors become more complex, laterally and medially projecting arbors remodel and clearly separate along the Xenopus neuropil.
To identify specific cellular actions of DSCAM in directing retinotopy in the tectum, we targeted the population of RGCs that preferentially express DSCAM to manipulate its expression when a majority of axons have already arborized in the tectum, but when medially and laterally projecting axons still overlap. For this, we electroporated a morpholino (MO) targeting Xenopus laevis Dscam mRNA to block translation and downregulate endogenous DSCAM levels in axons of ventral RGCs in tadpoles at stage 46, while also labeling dorsal axons with Alexa 488 dextran. This strategy allowed us to manipulate and visualize the innervation patterns and topographic organization of axon arbors in the neuropil rather than interfere with axon pathfinding or initial axon branching (27). As shown for control tadpoles, axons derived from ventral and dorsal RGCs were correctly sorted along the medial-lateral axis (Fig 7a, b), with ventral RGC axons predominantly arborizing in the medial portion of the neuropil and dorsal RGCs axons arborizing laterally. However, 48 hours after DSCAM MO injection, ventral RGC axon arbors were positioned more medially compared to controls (Fig 7b). To quantify this effect, we measured the area occupied by the axon arbors within the tectal neuropil; total arbor width from ventral RGCs injected with DSCAM MO was compared to that from ventral RGCs in tadpoles injected with control MO. The average arbor spread of axons positioned medially in tadpoles with DSCAM MO knockdown was significantly larger than controls (Controls 117.5 ± 9.73 μm, n = 6; DSCAM MO 150.2 ± 7.81 μm, n = 6, p = 0.0257, Fig 7c). Dorsal RGC axons labeled with Alexa 488 dextran projecting laterally within the tectum in either control MO or DSCAM MO treated tadpoles had the same arbor width independent of ventral RGC treatment (Controls 75.03 ± 8.723 μm, n = 6; DSCAM MO 78.85 ± 2.95 μm, n = 6, p = 0.6875, not significant, Fig 7d). As shown above, in stage 46 tadpoles there is a degree of overlap between medially-projecting ventral RGC axons (red marker) and laterally-projecting dorsal RGC axons (green marker) within the tectal neuropil (Fig 7b, e). At this stage, axons from ventral RGCs with DSCAM MO knockdown showed a significant reduction in lateral and medial arbor overlap compared to arbors in control MO treated tadpoles (Controls 22.02 ± 3.915 μm, n = 6; DSCAM MO 9.185 ± 2.193 μm, n = 6, *p = 0.0169, Fig 7e). When measuring the entire spread of the arbors along the medial to lateral axis for each group, tadpoles treated with DSCAM MO had RGC axons that occupied a larger territory within the neuropil (Controls 117.5 ± 9.733 μm width, n = 6; DSCAM MO 150.2 ± 7.808 μm, n = 6, *p = 0.0257, Fig 7f). When expressed as percentage overlap per total arbor territory in width the reduction in overlap for ventral axons with DSCAM MO knockdown was also significantly different from controls (Controls 18.82 ± 2.685 %, n = 6; DSCAM MO 6.135 ± 1.482 %, n = 6, **p = 0.0019, Fig 7g). Together, these findings suggest that changes in DSCAM expression in ventral RGC axons affect their projection patterns acting at the target where an increase in segregation of medial and lateral axons is observed in response to lowered endogenous DSCAM levels.
Dendritic Localization of DSCAM in Post-synaptic Tectal Neurons
Our previous work showed that downregulation of DSCAM expression in single RGCs interferes with axon growth and branching at the target, indicating that endogenous DSCAM acts as permissive cue that facilitates RGC axon growth. In contrast, single-cell downregulation or overexpression of DSCAM in tectal neurons showed that DSCAM acts as a restrictive cue to regulate the size and complexity of their dendritic arbors (27). Thus, in addition to RGCs, DSCAM can differentially influence postsynaptic neurons in the Xenopus visual system. Indeed, punctate DSCAM immunoreactivity can be detected not only within the Xenopus retina but also surrounding cell bodies in the tectum as well as in the tectal neuropil in unpermeabilized tissues (Fig 8a, b). To further differentiate DSCAM expression in tectal neurons, we electroporated embryos with a GFP plasmid, and at stage 45, tadpoles with isolated or small clusters of GFP-expressing neurons were fixed and immunostained for DSCAM. We found that DSCAM is not only expressed along the cell body surface of tectal neurons (white circle, Fig 8a), as previously shown (27), but is also expressed along primary dendrites and dendritic branches of tectal neurons (white arrows Fig 8a). Analysis of DSCAM immunostained tissues further revealed a unique pattern of expression, strongly labeling thin processes within the tectal neuropil (Figs 8 a, b; arrowheads). In some tadpoles, the random transfection and expression of GFP within brain neurons revealed strong DSCAM immunoreactivity along the primary processes of GFP-expressing cells that were positioned within the neuropil (Fig 8b). The identity of these cells in Xenopus is unknown, but they share similar morphology and features to tegmental projection neurons characterized in id2b transgenic zebrafish larvae that are found exclusively in the neuropil and have a prominent primary process that protrudes apically (34). Further experiments are needed to confirm their identity in Xenopus and potential roles for DSCAM in these neurons.