Bilateral visual projections exist in non-teleost bony fish and predate the emergence of tetrapods.

In most vertebrates, camera-style eyes contain retinal ganglion cell neurons that project to visual centers on both sides of the brain. However, in fish, ganglion cells were thought to innervate only the contralateral side, suggesting that bilateral visual projections appeared in tetrapods. Here we show that bilateral visual projections exist in non-teleost fishes and that the appearance of ipsilateral projections does not correlate with terrestrial transition or predatory behavior. We also report that the developmental program that specifies visual system laterality differs between fishes and mammals, as the Zic2 transcription factor, which specifies ipsilateral retinal ganglion cells in tetrapods, appears to be absent from fish ganglion cells. However, overexpression of human ZIC2 induces ipsilateral visual projections in zebrafish. Therefore, the existence of bilateral visual projections likely preceded the emergence of binocular vision in tetrapods.

Revisiting the role of Dcc in visual system development with a novel eye clearing method.

The Deleted in Colorectal Carcinoma (Dcc) receptor plays a critical role in optic nerve development. Whilst Dcc is expressed postnatally in the eye, its function remains unknown as Dcc knockouts die at birth. To circumvent this drawback, we generated an eye-specific Dcc mutant. To study the organization of the retina and visual projections in these mice, we also established EyeDISCO, a novel tissue clearing protocol that removes melanin allowing 3D imaging of whole eyes and visual pathways. We show that in the absence of Dcc, some ganglion cell axons stalled at the optic disc, whereas others perforated the retina, separating photoreceptors from the retinal pigment epithelium. A subset of visual axons entered the CNS, but these projections are perturbed. Moreover, Dcc-deficient retinas displayed a massive postnatal loss of retinal ganglion cells and a large fraction of photoreceptors. Thus, Dcc is essential for the development and maintenance of the retina.

Neurogenesis and Specification of Retinal Ganglion Cells.

Across all species, retinal ganglion cells (RGCs) are the first retinal neurons generated during development, followed by the other retinal cell types. How are retinal progenitor cells (RPCs) able to produce these cell types in a specific and timely order? Here, we will review the different models of retinal neurogenesis proposed over the last decades as well as the extrinsic and intrinsic factors controlling it. We will then focus on the molecular mechanisms, especially the cascade of transcription factors that regulate, more specifically, RGC fate. We will also comment on the recent discovery that the ciliary marginal zone is a new stem cell niche in mice contributing to retinal neurogenesis, especially to the generation of ipsilateral RGCs. Furthermore, RGCs are composed of many different subtypes that are anatomically, physiologically, functionally, and molecularly defined. We will summarize the different classifications of RGC subtypes and will recapitulate the specification of some of them and describe how a genetic disease such as albinism affects neurogenesis, resulting in profound visual deficits.

PlexinA2 and Sema6A are required for retinal progenitor cell migration.

In the vertebrate retina six types of neurons and one glial cell type are generated from multipotent retinal progenitor cells (RPCs) whose proliferation and differentiation are regulated by intrinsic and extrinsic factors. RPCs proliferate undergoing interkinetic nuclear migration within the neuroblastic layer, with their nuclei moving up and down along the apico-basal axis. Moreover, they only differentiate and therefore exit the cell cycle at the apical side of the neuroblastic layer. Sema6A and its receptors PlexinA4 and PlexinA2 control lamina stratification of the inner plexiform layer in the mouse retina. Nevertheless, their function in earlier developmental stages is still unknown. Here, we analyzed the embryonic retina of PlexinA2 and Sema6A knockout mice. Using time-lapse videomicroscopy we provide evidence that Sema6A/PlexinA2 signaling participates to interkinetic nuclear migration of RPCs around birth. When disrupted, RPCs migration is blocked at the apical side of the neuroblastic layer. This is the first evidence supporting a role for transmembrane molecules in the regulation of interkinetic nuclear migration in the mouse retina.

Development of retinal layers.

A noticeable characteristic of nervous systems is the arrangement of synapses into distinct layers. Such laminae are fundamental for the spatial organisation of synaptic connections transmitting different kinds of information. A major example of this is the inner plexiform layer (IPL) of the vertebrate retina, which is subdivided into at least ten sublayers. Another noticeable characteristic of these retina layers is that neurons are displayed in the horizontal plane in a non-random array termed as mosaic patterning. Recent studies of vertebrate and invertebrate systems have identified molecules that mediate these interactions. Here, we review the last mechanisms and molecules mediating retinal layering.

Guidance-cue control of horizontal cell morphology, lamination, and synapse formation in the mammalian outer retina.

In the vertebrate retina, neuronal circuitry required for visual perception is organized within specific laminae. Photoreceptors convey external visual information to bipolar and horizontal cells at triad ribbon synapses established within the outer plexiform layer (OPL), initiating retinal visual processing. However, the molecular mechanisms that organize these three classes of neuronal processes within the OPL, thereby ensuring appropriate ribbon synapse formation, remain largely unknown. Here we show that mice with null mutations in Sema6A or PlexinA4 (PlexA4) exhibit a pronounced defect in OPL stratification of horizontal cell axons without any apparent deficits in bipolar cell dendrite or photoreceptor axon targeting. Furthermore, these mutant horizontal cells exhibit aberrant dendritic arborization and reduced dendritic self-avoidance within the OPL. Ultrastructural analysis shows that the horizontal cell contribution to rod ribbon synapse formation in PlexA4⁻/⁻ retinas is disrupted. These findings define molecular components required for outer retina lamination and ribbon synapse formation.

Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs(∗)57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated.

Slit2 activity in the migration of guidepost neurons shapes thalamic projections during development and evolution.

How brain connectivity has evolved to integrate the mammalian-specific neocortex remains largely unknown. Here, we address how dorsal thalamic axons, which constitute the main input to the neocortex, are directed internally to their evolutionary novel target in mammals, though they follow an external path to other targets in reptiles and birds. Using comparative studies and functional experiments in chick, we show that local species-specific differences in the migration of previously identified "corridor" guidepost neurons control the opening of a mammalian thalamocortical route. Using in vivo and ex vivo experiments in mice, we further demonstrate that the midline repellent Slit2 orients migration of corridor neurons and thereby switches thalamic axons from an external to a mammalian-specific internal path. Our study reveals that subtle differences in the migration of conserved intermediate target neurons trigger large-scale changes in thalamic connectivity, and opens perspectives on Slit functions and the evolution of brain wiring.

Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina.

In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.

Chemoattractive activity of sonic hedgehog in the adult subventricular zone modulates the number of neural precursors reaching the olfactory bulb.

The adult subventricular zone (SVZ) supports neural stem cell self-renewal and differentiation and continually gives rise to new neurons throughout adult life. The mechanisms orienting the migration of neuroblasts from the SVZ to the olfactory bulb (OB) via the rostral migratory stream (RMS) have been extensively studied, but factors controlling neuroblast exit from the SVZ remain poorly explored. The morphogen Sonic Hedgehog (Shh) displays proliferative and survival activities toward neural stem cells and is an axonal chemoattractant implicated in guidance of commissural axons during development. We identify here the presence of Shh protein in SVZ extracts and in the cerebrospinal fluid of adult mice, and we demonstrate that migrating neuroblasts in the SVZ and RMS express the Shh receptor Patched. We show that Shh displays a chemoattractive activity in vitro on SVZ-derived neuronal progenitors, an effect blocked by Cur61414, a Smoothened antagonist. Interestingly, Shh-expressing cells grafted above the RMS of adult mice exert a chemoattractive activity on migrating neuroblasts in vivo, thus inducing their accumulation and deviation from their normal migratory pathway. Furthermore, the adenoviral transfer of Shh into the lateral ventricle or the blocking of Shh present in the SVZ of adult mice using its physiological antagonist Hedgehog interacting protein or neutralizing Shh antibodies provides in vivo evidence that Shh can retain SVZ-derived neuroblasts. The ability to modulate the number of neuroblasts leaving the SVZ and reaching the OB through the chemoattractive activity of Shh suggests a novel degree of plasticity in cell migration of this adult stem cell niche.

Molecular mechanisms controlling midline crossing by precerebellar neurons.

Precerebellar neurons of the inferior olive (IO) and lateral reticular nucleus (LRN) migrate tangentially from the rhombic lip toward the floor plate following parallel pathways. This process is thought to involve netrin-1 attraction. However, whereas the cell bodies of LRN neurons cross the midline, IO neurons are unable to do so. In many systems and species, axon guidance and cell migration at the midline are controlled by Slits and their receptor Robos. We showed previously that precerebellar axons and neurons do not cross the midline in the absence of the Robo3 receptor. To determine whether this signaling by Slits and the two other Robo receptors, Robo1 and Robo2, also regulates precerebellar neuron behavior at the floor plate, we studied the phenotype of Slit1/2 and Robo1/2/3 compound mutants. Our results showed that many IO neurons can cross the midline in absence of Slit1/2 or Robo1/2, supporting a role for midline repellents in guiding precerebellar neurons. We also show that these molecules control the development of the lamellation of the inferior olivary complex. Last, the analysis of Robo1/2/3 triple mutants suggests that Robo3 inhibits Robo1/2 repulsion in precrossing LRN axons but not in IO axons in which it has a dominant and distinct function.

Robos and slits control the pathfinding and targeting of mouse olfactory sensory axons.

Odorants are detected by olfactory receptor neurons (ORNs) located in the olfactory epithelium. In mice, ORNs expressing the same odorant receptor (OR) project to a single glomerulus out of 1800 in the olfactory bulb (OB). It has been proposed that OR-derived cAMP signals guide ORN axons to their glomeruli rather than OR themselves. Recently, it has also been shown that the axon guidance molecule Slit1 and its receptor Robo2 control the dorsoventral segregation of ORN axons as they are projecting to the OB. We have analyzed the development of olfactory projections in Slit1/Slit2 and Robo1/Robo2 single and double mutants. We show that in Robo1-/-;Robo2-/- mice, most ORN axons fail to enter the OB and instead project caudally into the diencephalon. Moreover, in these mice, ORN axons expressing the same OR project to several glomeruli at ectopic positions. Thus, Slit1, Slit2, Robo1, and Robo2 cooperate to control the convergence of ORN axons to the OB and the precise targeting of ORN axons to specific glomeruli.

Robo1 and robo2 control the development of the lateral olfactory tract.

The development of olfactory bulb projections that form the lateral olfactory tract (LOT) is still poorly understood. It is known that the septum secretes Slit1 and Slit2 which repel olfactory axons in vitro and that in Slit1-/-;Slit2-/- mutant mice, the LOT is profoundly disrupted. However, the involvement of Slit receptors, the roundabout (Robo) proteins, in guiding LOT axons has not been demonstrated. We show here that both Robo1 and Robo2 receptors are expressed on early developing LOT axons, but that only Robo2 is present at later developmental stages. Olfactory bulb axons from Robo1-/-;Robo2-/- double-mutant mice are not repelled by Slit in vitro. The LOT develops normally in Robo1-/- mice, but is completely disorganized in Robo2-/- and Robo1-/-;Robo2-/- double-mutant embryos, with many LOT axons spreading along the ventral surface of the telencephalon. Finally, the position of lot1-expressing cells, which have been proposed to be the LOT guidepost cells, appears unaffected in Slit1-/-;Slit2-/- mice and in Robo1-/-;Robo2-/- mice. Together, our results indicate that Robo1 and Robo2 directly mediate the repulsive activity of Slit receptors on LOT axons, and are required for normal guidance of these axons in vivo.

Under the eye of Nr-CAM.

Binocular vision relies upon the existence of contralateral and ispilateral projections from retinal ganglion cells. Contacts between visual axons and optic chiasm cells are critical for the sorting of crossed and uncrossed projections during development. In this issue of Neuron, a study by Williams et al. shows that the cell adhesion molecule Nr-CAM facilitates/promotes the decussation of contralateral axons across the chiasm.

Multiple roles for slits in the control of cell migration in the rostral migratory stream.

The subventricular zone (SVZ) contains undifferentiated cells, which proliferate and generate olfactory bulb (OB) interneurons. Throughout life, these cells leave the SVZ and migrate along the rostral migratory stream (RMS) to the OB where they differentiate. In vitro, the septum and the choroid plexus (CP) secrete repulsive factors that could orient the migration of OB precursors. Slit1 and Slit2, two known chemorepellents for developing axons, can mimic this effect. We show here that the Slit receptors Robo2 and Robo3/Rig-1 are expressed in the SVZ and the RMS and that Slit1 and Slit2 are still present in the adult septum. Using Slit1/2-deficient mice, we found that Slit1 and Slit2 are responsible for both the septum and the CP repulsive activity in vitro. In adult mice lacking Slit1, small chains of SVZ-derived cells migrate caudally into the corpus callosum, supporting a role for Slits in orienting the migration of SVZ cells. Surprisingly, in adult mice, Slit1 was also expressed by type A and type C cells in the SVZ and RMS, suggesting that Slit1 could act cell autonomously. This hypothesis was tested using cultures of SVZ explants or isolated neurospheres from Slit1-/- or Slit1+/- mice. In both types of cultures, the migration of SVZ cells was altered in the absence of Slit1. This suggests that the regulation of the migration of OB precursors by Slit proteins is complex and not limited to repulsion.

Slit1 and slit2 proteins control the development of the lateral olfactory tract.

The development of olfactory bulb projections that form the lateral olfactory tract (LOT) is still poorly understood. The septum and the olfactory cortex have been shown to secrete diffusible factors repelling olfactory axons in vitro and are likely to cause the axons to avoid the septum region in vivo. Slit2, a member of the Slit gene family, has been proposed to be this septal factor based on its expression in the embryonic septum and its ability to repel and collapse olfactory axons. However, this issue is still controversial, and recent in vitro studies have questioned the role of the septum and Slit proteins in organizing LOT projections. We therefore decided to examine directly the role of Slit proteins in mediating olfactory axon guidance in vivo using mice with targeted deletions in the Slit1 and Slit2 genes. When olfactory bulb explants are cocultured with septum from Slit1- and/or Slit2-deficient mice, the septum repulsive activity for olfactory bulb axons is progressively abolished in a gene dose-dependent manner. Anterograde tracing of olfactory bulb axons showed that the LOT develops normally in Slit1 or Slit2 single-deficient mice but is completely disorganized in Slit1/Slit2 double-deficient embryos, with many axons reaching the midline and entering the septum region. Therefore, our study showed that the septum chemorepellent is a combination of Slit1 and Slit2 and that these molecules play a significant role in olfactory bulb axon guidance in vivo.

Role of Slit proteins in the vertebrate brain.

Diffusible chemorepellents play a major role in guiding developing axons towards their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptors and their secreted ligand Slits. Three distinct slit genes (slit1, slit2 and slit3) and three distinct robo genes (robo1, robo2 and rig-1) have been cloned in mammals. In collagen gel co-cultures, Slit1 and Slit2 can repel and collapse olfactory axons. However, there is also some positive effect associated with Slits, as Slit2 stimulates the formation of axon collateral branches by NGF-responsive neurons of the dorsal root ganglia (DRG). Slit2 is a large ECM glycoproteins of about 200 kD, which is proteolytically processed into 140 kD N-terminal and 55-60 kD C-terminal fragments. Slit2 cleavage fragments appear to have different cell association characteristics, with the smaller C-terminal fragment being more diffusible and the larger N-terminal and uncleaved fragments being more tightly cell associated. This suggested that the different fragments might have different functional activities in vivo. We have begun to explore these questions by engineering mutant and truncated versions of hSlit2 representing the two cleavage fragments, N- and C-, and the uncleavable molecule and examining the activities of these mutants in binding and functional assays. We found that an axon's response to Slit2 is not absolute, but rather is reflective of the context in which the protein is encountered.

Spatiotemporal expression patterns of slit and robo genes in the rat brain.

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a repulsive guidance system that prevents inappropriate axons from crossing the central nervous system midline; this repulsive system is mediated by the secreted extracellular matrix protein Slit and its receptors Roundabout (Robo). Three distinct slit genes (slit1, slit2, and slit3) and three distinct robo genes (robo1, robo2, rig-1) have been cloned in mammals. However, to date, only Robo1 and Robo2 have been shown to be receptors for Slits. In rodents, Slits have been shown to function as chemorepellents for several classes of axons and migrating neurons. In addition, Slit can also stimulate the formation of axonal branches by some sensory axons. To identify Slit-responsive neurons and to help analyze Slit function, we have studied, by in situ hybridization, the expression pattern of slits and their receptors robo1 and robo2, in the rat central nervous system from embryonic stages to adult age. We found that their expression patterns are very dynamic: in most regions, slit and robo are expressed in a complementary pattern, and their expression is up-regulated postnatally. Our study confirms the potential role of these molecules in axonal pathfinding and neuronal migration. However, the persistence of robo and slit expression suggests that the couple slit/robo may also have an important function in the adult brain.