Differential Expression of DSCAM Guides the Patterning of Retinal Axons Along Their Path and at Their Target in the Developing Xenopus Visual System

Background The Xenopus retinotectal circuit is organized topographically, where the dorsal-ventral axis of the retina maps respectively on to the ventral-dorsal axis of the tectum; axons from the nasal-temporal axis of the retina project respectively to the caudal-rostral axis of the tectum. Studies throughout the last two decades have shown that mechanisms involving molecular recognition of proper termination domains are at work guiding topographic organization. Such studies have shown that graded distribution of molecular cues is important for topographic mapping. However, the molecular cues organizing topography along the developing optic nerve, and as retinal axons cross the chiasm and navigate towards their target in the tectum, remain unknown. Down syndrome cell adhesion molecule (DSCAM) has been characterized as a key molecule in axon guidance, making it a strong candidate involved in the topographic organization of retinal fibers along the optic path.Methods Using a combination of whole-brain clearing and immunohistochemistry staining techniques we characterized DSCAM expression and the projection of ventral and dorsal retinal fibers starting from the eye, followed to the optic nerve into the chiasm, and into the terminal target in the optic tectum in Xenopus laevis tadpoles. We also assessed the effects of DSCAM on the establishment of retinotopic maps through spatially and temporally targeted DSCAM knockdown on retinal ganglion cells (RGCs) with axons innervating the optic tectum. Results Highest expression of DSCAM was localized to the ventral posterior region of the optic nerve and chiasm; this expression pattern coincides with ventral fibers derived from ventral RGCs. Downregulating DSCAM levels affected the segregation and proper sorting of medial axon fibers, derived from ventral RGCs, within the tectal neuropil, indicating that DSCAM plays a role in retinotopic organization. ConclusionThese findings together with the observation that DSCAM immunoreactivity accumulates on the primary dendrites of tectal neurons indicates that DSCAM exerts multiple roles in coordinating retinotopic order and connectivity in the developing vertebrate visual system.

Activity-dependent refinement of nasal retinal projections drives topographic map sharpening in the teleost visual system

ABSTRACTTopographic maps in the brain are essential for processing information. Yet, our understanding of topographic mapping has remained limited by our inability to observe maps forming and refining directly in vivo. Here, we used Cre-mediated recombination of a new colorswitch reporter in zebrafish to generate the first transgenic model allowing the dynamic analysis of retinotopic mapping in vivo. We found that the antero-posterior retinotopic map forms early but remains dynamic, with nasal and temporal retinal axons expanding their projection domains over time. Nasal projections initially arborize in the anterior tectum but progressively refine their projection domain to the posterior tectum in an activity-dependent manner. This activity-dependent refinement drives retinotopic map sharpening along the antero-posterior axis. Altogether, our study provides the first analysis of a topographic map maturing in real-time in a live animal and opens new strategies for dissecting the intricate mechanisms of precise topographic mapping in vertebrates.

A Zic2-regulated switch in a noncanonical Wnt/βcatenin pathway is essential for the formation of bilateral circuits

The Wnt pathway is involved in a wide array of biological processes during development and is deregulated in many pathological scenarios. In neurons, Wnt proteins promote both axon extension and repulsion, but the molecular mechanisms underlying these opposing axonal responses are unknown. Here, we show that Wnt5a is expressed at the optic chiasm midline and promotes the crossing of retinal axons by triggering an alternative Wnt pathway that depends on the accumulation of βcatenin but does not activate the canonical pathway. In ipsilateral neurons, the transcription factor Zic2 switches this alternative Wnt pathway by regulating the expression of a set of Wnt receptors and intracellular proteins. In combination with this alternative Wnt pathway, the asymmetric activation of EphB1 receptors at the midline phosphorylates βcatenin and elicits a repulsive response. This alternative Wnt pathway and its Zic2-triggered switch may operate in other contexts that require a two-way response to Wnt ligands.

Diverse GABAergic neurons organize into subtype-specific sublaminae in the ventral lateral geniculate nucleus

In the visual system, retinal axons convey visual information from the outside world to dozens of distinct retinorecipient brain regions and organize that information at several levels, including either at the level of retinal afferents, cytoarchitecture of intrinsic retinorecipient neurons, or a combination of the two. Two major retinorecipient nuclei which are densely innervated by retinal axons are the dorsal lateral geniculate nucleus (dLGN), which is important for classical image-forming vision, and ventral LGN (vLGN), which is associated with non-image-forming vision. The neurochemistry, cytoarchitecture, and retinothalamic connectivity in vLGN remain unresolved, raising fundamental questions of how it receives and processes visual information. To shed light on these important questions, we labeled neurons in vLGN with canonical and novel cell type-specific markers and studied their spatial distribution and morphoelectric properties. Not only did we find a high percentage of cells in vLGN to be GABAergic, we discovered transcriptomically distinct GABAergic cell types reside in the two major laminae of vLGN, the retinorecipient, external vLGN (vLGNe) and the non-retinorecipient, internal vLGN (vLGNi). Within vLGNe, we identified transcriptionally distinct subtypes of GABAergic cells that are distributed into four adjacent sublaminae. Using trans-synaptic viral tracing and in vitro electrophysiology, we found cells in each these vLGNe sublaminae receive monosynaptic inputs from the retina. These results not only identify novel subtypes of GABAergic cells in vLGN, they suggest the subtype-specific laminar distribution of retinorecipient cells in vLGNe may be important for receiving, processing, and transmitting light-derived signals in parallel channels of the subcortical visual system.Graphical abstract.The vLGN is organized into subtype-specific sublaminae which receive visual inputThe ventral lateral geniculate nucleus (vLGN) is part of the visual thalamus. It can broadly be separated into two structural domains or laminae, the external vLGNe (which receives retinal input) and the internal vLGNi (receives no retinal input). In this study, we describe subtypes of transcriptomically distinct GABAergic neurons that populate the vLGN and organize into discrete, adjacent sublaminae in the vLGNe. Taken together, our results show four subtype-specific sublaminae of retinorecipient neurons in vLGNe.

Retinal outputs depend on behavioural state

The operating mode of the visual system depends on behavioural states such as arousal1,2. This dependence is seen both in primary visual cortex3–7 (V1) and in subcortical brain structures receiving direct retinal input4,8. Here we show that this effect arises as early as in the output of the retina. We first measured activity in a region that receives retinal projections9, the superficial superior colliculus (sSC), and found that this activity strongly depended on behavioural state. This modulation was not mediated by feedback inputs from V1 as it was immune to V1 inactivation. We then used Neuropixels probes10 to record activity in the optic tract, and we found some retinal axons whose activity significantly varied with arousal, even in darkness. To characterize these effects on a larger sample of retinal outputs, we imaged the activity of retinal boutons11,12 in sSC during behaviour using a calcium indicator. The activity of these boutons correlated with arousal as strongly as that of sSC neurons, and this correlation persisted also during darkness. These results reveal a novel property of retinal function in mice, which could be observed only during behaviour: retinal outputs are modulated by behavioural state before they reach the rest of the brain.

Nell2 regulates the contralateral-versus-ipsilateral visual projection as a layer-specific positional cue

SUMMARY STATEMENTNell2 is an ipsilateral layer-specific axon guidance cue in the visual thalamus and contributes to establishment of the eye-specific retinogeniculate projection by specifically inhibiting contralateral retinal axons.ABSTRACTIn mammals with binocular vision, retinal ganglion cell (RGC) axons from each eye project to eye-specific layers in the contralateral and ipsilateral dorsal lateral geniculate nucleus (dLGN). Although layer-specific axon guidance cues that discriminate contralateral and ipsilateral RGC axons have long been postulated as a key mechanism for development of the eye-specificretinogeniculate projection, the molecular nature of such cues has remained elusive. Here we show that the extracellular glycoprotein Nell2 (also known as Nel) is expressed in the dorsomedial region of the dLGN, which corresponds to the layer receiving ipsilateral RGC axons. In Nell2 mutant mice, contralateral RGC axons invaded the ipsilateral layer of the dLGN, and ipsilateral axons terminated in partially fragmented patches, forming a mosaic pattern of contralateral and ipsilateral axon termination zones. In vitro, Nell2 exerted inhibitory effects on contralateral, but not ipsilateral, RGC axons. These results provide evidence that Nell2 acts as a layer-specific positional label in the dLGN that discriminates contralateral and ipsilateral RGC axons, and that it plays essential roles in establishment of the eye-specific projection patterns in the retinogeniculate system.

CRMP2 and CRMP4 Are Differentially Required for Axon Guidance and Growth in Zebrafish Retinal Neurons

Axons are directed to their correct targets by guidance cues during neurodevelopment. Many axon guidance cues have been discovered; however, much less known is about how the growth cones transduce the extracellular guidance cues to intracellular responses. Collapsin response mediator proteins (CRMPs) are a family of intracellular proteins that have been found to mediate growth cone behavior in vitro; however, their roles in vivo in axon development are much less explored. In zebrafish embryos, we find that CRMP2 and CRMP4 are expressed in the retinal ganglion cell layer when retinal axons are crossing the midline. Knocking down CRMP2 causes reduced elongation and premature termination of the retinal axons, while knocking down CRMP4 results in ipsilateral misprojections of retinal axons that would normally project to the contralateral brain. Furthermore, CRMP4 synchronizes with neuropilin 1 in retinal axon guidance, suggesting that CRMP4 might mediate the semaphorin/neuropilin signaling pathway. These results demonstrate that CRMP2 and CRMP4 function differentially in axon development in vivo.