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 membrane of neurons in the RGC layer within the retina and neurons in the optic tectum in Xenopus tadpoles at stage 45 [23]. Without permeabilization, punctate DSCAM immunoreactivity was localized to the tectal neuropil where retinal axons and tectal neuron 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 [23]) 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 immunoreactivity was highly localized to the ventral region of the optic nerve (Fig. 1a, b). Fluorescence intensity for both DSCAM and 3A10 immunoreactivity was 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 normalized and plotted fluorescence intensity on a graph with the x-axis representing regions along the ventral to dorsal portions of the optic nerve (Fig. 1d; n = 4 tadpoles). This analysis quantitatively confirmed a high-ventral to low-dorsal graded pattern of DSCAM expression. In contrast, 3A10 immunoreactivity strongly localized to the dorsal region of the optic nerve (border noted by white dotted line on Fig. 1b) with a lower intensity signal observed along the ventral side. Quantitatively, 3A10 immunofluorescence was lower ventral and higher dorsal (Fig. 1d), a distribution that appeared to be an inverse of the high-ventral to low-dorsal DSCAM immunoreactivity pattern. Similarly, 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 gradually decreased towards the more dorsal areas of the optic chiasm (white dotted line, Fig. 1c, e), while 3A10 immunoreactivity was higher dorsally along the optic chiasm (Fig. 1c, e). DSCAM immunoreactivity became less distinguishable in axon fibers projecting contralaterally along the optic tract past the chiasm (arrow, Fig. 1c).
Having encountered a differential distribution of DSCAM along the ventrodorsal axis of the optic nerve and chiasm, we further characterized DSCAM expression along the posterior-anterior 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 DSCAM and 3A10 immunoreactivity co-localized along a number of fibers both in the optic nerve and chiasm (white arrows, Fig. 2a), confirming that the DSCAM immunostaining identified RGC axon fibers. These results suggest that a subpopulation of RGC axon fibers differentially relies 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 [25]. Cleared tissue samples of stage 45 to 46 tadpoles were immunostained with DSCAM and 3A10 antibodies (Fig. 3). This technique revealed that the optic nerve (solid white arrowhead, Fig. 3a), as well as sensory and motor cranial nerves throughout the tadpole head were immunostained by the 3A10 antibody (Fig. 3a). Individual confocal planes of cleared brain tissues imaged ventrally showed that DSCAM immunoreactivity was higher posteriorly along the optic nerve as it enters the optic chiasm (arrow, Fig. 3b). At the optic tectal neuropil, strongest DSCAM immunoreactivity coincided with axon terminals labeled with the 3A10 antibody (empty white arrowheads at the tectum, Fig. 3a). When we examined individual horizontal z-stacks, we observed that within the neuropil, DSCAM immunoreactivity was higher at the level where RGC axons terminate as revealed by the 3A10 co-immunostaining (solid white and yellow arrowheads; Fig. 3c, left and middle panels), and lower at fasciculated axon bundle tracts as they enter the neuropil (empty white and yellow arrowheads, Fig. 3c, middle and right panels). Viewing single confocal orthogonal planes of cleared, dissected brain tissues confirmed that strong DSCAM immunoreactivity localized to the area of the neuropil with the 3A10 retinal axon marker (solid white and yellow arrows, Fig. 3d). Single plane analysis of high magnification confocal z-stacks also revealed DSCAM punctate staining on 3A10-stained growth cones in axons terminals (Fig. 3e). When compared to the area devoid of retinal fibers, quantitatively DSCAM fluorescence intensity was significantly higher within the area of the tectal neuropil where 3A10-immunolabeled RGC axon terminals localize (Fig. 3f). DSCAM immunoreactivity was also higher within the area where RGC axons terminate when compared to the more anterior optic tract where the 3A10-immunostained axon fibers path-find (Fig. 3f).
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 in cryostat sections immunostained with DSCAM and the 3A10 antibodies (Fig. 4). As previously shown [23], DSCAM immunoreactivity was observed in the ganglion cell layer (GCL), inner plexiform layer (IPL) and inner nuclear layer (INL) of the Xenopus retina, with punctate DSCAM expression found around cell bodies within the GCL (Fig. 4a). Because no differential expression of DSCAM could be discerned within the layers of the retina between the dorsal or ventral axis (Fig. 4a, see also [23]), it is possible that DSCAM function along optic axon nerve bundles and axon terminals is separate or independent from its function within the local retinal circuit [22, 23, 29,30,31]. In the retina, 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. 4a-c) indicating that subsets of RGCs differentially express the neurofilament-associated proteins recognized by the 3A10 antibody. Analysis of confocal sections of retinas co-immunostained with DSCAM and 3A10 antibodies showed that some axon fibers exiting the eye along the optic fiber layer (Fig. 4c; arrow) and the optic nerve head (Fig. 4c, box, Fig. 4d) were immunopositive for both DSCAM and 3A10 (Fig. 4d; empty arrowheads), although stronger DSCAM immunoreactivity localized to axon fibers with weaker 3A10 immunoreactivity (Fig. 4e; empty arrowheads). However, a number of 3A10 positive fibers did not stain for DSCAM (white arrowheads; Fig. 4d, f). Together, these observations show a differential pattern of expression of DSCAM by RGC axons as they exit the eye and reveal that 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 path 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 and/or at their target [32]. 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. 5a). 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. 5b, c). Although fluorescently tagged morpholinos do not stain and reveal the entire complexity of RGC axon terminal arbors, their transport along the axons to the tip of the terminals served as a reliable marker to topographically target and label RGCs and their axons. 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. 5c, 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 after crossing the chiasm (Fig. 5d, 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. 5f). 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. 5g) in agreement with previous studies [33, 34]. Thus, our analysis of the topographic organization of dorsal and ventral RGC axons as they travel from the optic nerve to their target complements our immunohistochemical data and 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 towards the tectum (Figs. 1b, c and 3b).
Analysis of axon terminals along the lateral-medial axis (as depicted schematically in Fig. 6a), showed that ventral RGC axons (labeled with lissamine-tagged MO) innervate the tectum medially, while dorsal RGC axons (labeled with Alexa 488 dextran) travel more laterally, as shown for other species [9, 35]. Indeed, ventral and dorsal RGC axons from tadpoles injected at stage 46 and imaged 48 h later (see Fig. 6b) showed correct topographic mapping but with a consistent degree of arbor overlap as shown in Fig. 6d. When retinal neurons were labeled at a later stage, at stage 47 and imaged 48 h after, medial arbors were visibly separated from lateral arbors (Fig. 6c). 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 at the time 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 [23]. As shown for control tadpoles, axons derived from ventral and dorsal RGCs were correctly sorted along the medial–lateral axis (Fig. 6d), with ventral RGC axons predominantly arborizing in the medial portion of the neuropil and dorsal RGCs axons arborizing laterally. However, 48 h after DSCAM MO injection, ventral RGC axon arbors seemed to be positioned more medially compared to controls (Fig. 6d). To quantify this effect, we measured the area occupied by the axon arbors within the tectal neuropil; total arbor area (in pixels) from ventral RGCs injected with DSCAM MO was compared to that from ventral RGCs in sibling tadpoles injected with control MO. The average arbor spread of axons positioned medially in tadpoles with DSCAM MO knockdown was not significantly different from controls (Fig. 6f). Dorsal RGC axons labeled with Alexa 488 dextran projecting laterally within the tectum in either control MO or DSCAM MO treated tadpoles also occupied a similar area independent of ventral RGC treatment (Fig. 6f). As shown above, in stage 46 tadpoles there is a degree of overlap between medially-projecting ventral RGC axons (red fluorescence) and laterally-projecting dorsal RGC axons (green fluorescence) within the tectal neuropil (Fig. 6d). When calculating the area of overlap occupied by dorsal (Alexa 488-dextran) and ventral RGC (lissamine-tagged MO) axons in the same tadpoles (normalized to percent of total area), DSCAM MO knockdown showed a significant reduction in arbor overlap compared to the overlap of arbors in control MO treated tadpoles (Fig. 6e). This difference was significant when analyzing overlap in relation to the area extent of the medially projecting ventral RGC axons (red fluorescence; Control MO 47.50 ± 2.7%, n = 8; DSCAM MO 24.55 ± 2.5%, n = 8, p ≤ 0.0001), laterally projecting dorsal RGC axons (green fluorescence; Control MO 44.49 ± 4.3%; DSCAM MO 23.20 ± 2.3%, p = 0.0004) and the combined projection of medial and laterally projecting axons (red and green fluorescence; Control MO 24.04 ± 1.1%; DSCAM MO 11.87 ± 1.1%, p ≤ 0.0001). Together, these findings suggest that changes in DSCAM expression in ventral RGC axons affect their projection patterns 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 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 [23]. 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. 7a, see also [23]). Analysis of tissues further revealed a unique pattern DSCAM immunoreactivity, with the DSCAM antibody strongly labeling thin processes within the tectal neuropil (Fig. 7a, arrowheads). To further characterize DSCAM expression, we electroporated embryos with a GFP plasmid at low concentration to randomly label cells in the brain. At stage 45, tadpoles were screened for the presence of isolated or small clusters of GFP-expressing neurons and were fixed and immunostained for DSCAM. DSCAM immunoreactivity localized to cell bodies, primary dendrites and dendritic branches of GFP-expressing tectal neurons (white arrows Fig. 7a). The random transfection and expression of GFP within the brain also revealed strong DSCAM immunoreactivity on primary processes of GFP-expressing cells that were positioned within the neuropil (Fig. 7b). 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 [36]. Thus, these experiments confirm specific localization of DSCAM not only on RGC axons but also on tectal neurons and raise the possibility of additional potential roles for DSCAM in neurons within the neuropil.