Construction and reconstruction of brain circuits: normal and pathological axon guidance.

Perception of our environment entirely depends on the close interaction between the central and peripheral nervous system. In order to communicate each other, both systems must develop in parallel and in coordination. During development, axonal projections from the CNS as well as the PNS must extend over large distances to reach their appropriate target cells. To do so, they read and follow a series of axon guidance molecules. Interestingly, while these molecules play critical roles in guiding developing axons, they have also been shown to be critical in other major neurodevelopmental processes, such as the migration of cortical progenitors. Currently, a major hurdle for brain repair after injury or neurodegeneration is the absence of axonal regeneration in the mammalian CNS. By contrasts, PNS axons can regenerate. Many hypotheses have been put forward to explain this paradox but recent studies suggest that hacking neurodevelopmental mechanisms may be the key to promote CNS regeneration. Here we provide a seminar report written by trainees attending the second Flagship school held in Alpbach, Austria in September 2018 organized by the International Society for Neurochemistry (ISN) together with the Journal of Neurochemistry (JCN). This advanced school has brought together leaders in the fields of neurodevelopment and regeneration in order to discuss major keystones and future challenges in these respective fields.

Dynamic expression of p75NTR and Lingo-1 during development of mouse retinofugal pathway.

Our previous studies showed interaction of Nogo at the midline with its receptor (NgR) on optic axons plays a role in axon divergence at the mouse optic chiasm. Since NgR lacks a cytoplasmic domain, it needs transmembrane receptor partners for signal transduction. In this study, we examined whether the co-receptors of NgR, low-affinity neurotrophic receptor (p75NTR) and Lingo-1, are localized on axons in the mouse optic pathway. In the retina, p75NTR and Lingo-1 were observed on neuroepithelial cells at E13 and later on the retinal ganglion cells at E14 and E15. At the optic disc, p75NTR was observed on the retinal axons, whereas Lingo-1 was found on glial processes surrounding the axon fascicles. Both p75NTR and Lingo-1 were found on axons in the optic stalk, optic chiasm and optic tract. Furthermore, a transient expression of Lingo-1 was observed on the SSEA-1 positive chiasmatic neurons at E13, but not at later developmental stages. The presence of p75NTR and Lingo-1 on optic axons provides further supports to the contribution of Nogo/NgR signaling in axon divergence at the mouse optic chiasm.

Histamine involvement in visual development and adaptation.

PURPOSE: This study evaluated the level of histamine in the interaction between the environment and the visual system during lifespan development, exploring potential sex differences. METHODS: Male and female Wistar rats, reared in standard laboratory or enriched-environment cages from birth to prepuberty or adulthood, were sacrificed during the critical period for visual development at postnatal day (P) 25 (P25) or in adulthood at P90. Additionally, animals born in standard conditions were exposed to an enriched environment at P90 and sacrificed at P150. The optic chiasm and the visual cortex were dissected out and tissue histamine was quantified fluorophotometrically. Statistical analyses were performed by ANOVA. RESULTS: Histamine levels in the optic chiasm were higher in male than in female rats at all ages. Comparable sex differences in the visual cortex were observed only during prepuberty. Basal histamine content in the optic chiasm was higher in prepuberty and decreased in adulthood in a sex-independent manner. Exposure to an enriched environment decreased optic chiasm histamine levels in both sexes and resulted in no sex difference in the cortical histamine levels at any age. Increased amine levels were detected in the optic chiasm of female rats exposed to an enriched environment during adulthood. CONCLUSIONS: This study presents first evidence associating central histamine levels with the visual system development and environmental adaptation, thus providing the lead for the investigation of the hitherto elusive role of histamine in the regulation of visual processes. Furthermore, the findings challenge the impact of laboratory animal raising environments in developmental and behavioral studies.

Optic chiasm formation in planarian I: Cooperative netrin- and robo-mediated signals are required for the early stage of optic chiasm formation.

Freshwater planarians can regenerate a brain, including eyes, from the anterior blastema, and coordinately form an optic chiasm during eye and brain regeneration. To investigate the role of the netrin- and slit-signaling systems during optic chiasm formation, we cloned three receptor genes (Djunc5A, Djdcc and DjroboA) expressed in visual neurons and their ligand genes (DjnetB and Djslit) and analyzed their functions by RNA interference (RNAi). Although each of DjroboA(RNAi), Djunc5A(RNAi) and DjnetB(RNAi) showed a weak phenotype and Djslit(RNAi) showed a severe defect of eye formation, we did not observe any defect of crossing of visual axons over the midline among single knockdown planarians. However, among double knockdown planarians, some of DjnetB(RNAi);DjroboA(RNAi) and Djunc5A(RNAi);DjroboA(RNAi) showed complete disconnection between the visual axons from the two sides, suggesting that some combination of netrin- and robo-mediated signals may be required for crossing over the midline. Finally, we carefully investigated the distribution patterns of cells expressing DjNetB protein, DjnetB, and Djslit at the early stage of regeneration, and found that visual axons projected along a path sandwiched between DjNetB protein and Djslit-positive cells. These results suggest that two different collaborative or combinatory signals may be required for midline crossing at the early stage of chiasm formation during eye and brain regeneration.

Detrimental effects of cigarette smoke constituents on physiological development of extraocular and intraocular structures.

No investigation has yet been accomplished to screen the detrimental effects of cigarette smoke condensate (CSC) and total particular matter solution (TPMS) on embryonic development of extraocular and intraocular structures. In this report, chicken embryo assay was utilized to undermine diverse ocular pathologies produced by exposure of CSC and TPM. Extraocular anomalies triggered after exposure of CSC and TPMS include degeneration of optic chiasma, medial rectus muscle, and inflammatory lesions in forebrain. Histological investigations of CSC and TPMS-treated embryos also exposed delayed differentiation of photoreceptor layer, degeneration of retinal ganglion and nerve cell layer. In addition, corneal thickness, deterioration and complete loss of hyaloid vasculature were observed. Extraocular and intraocular regions of TPMS-treated embryos also revealed widespread hemorrhages in the entire cephalic, optic disc, ganglion cell layer and vitreous humor area. The findings of our experiment demonstrate, for the first time, that exposure to CSC and TPMS is hazardous for developing embryos and it has potential detrimental effects on several underlying events of ocular development. Moreover, it was also intriguing that toxicity profile of TMP was much more higher than CSC with more profound detrimental effects on ocular development.

The growth-inhibitory protein Nogo is involved in midline routing of axons in the mouse optic chiasm.

We have investigated the role of Nogo, a protein that inhibits regenerating axons in the adult central nervous system, on axon guidance in the developing optic chiasm of mouse embryos. Nogo protein is expressed by radial glia in the midline within the optic chiasm where uncrossed axons turn, and the Nogo receptor (NgR) is expressed on retinal neurites and growth cones. In vitro neurite outgrowth from both dorsonasal and ventrotemporal retina was inhibited by Nogo protein, and this inhibition was abolished by blocking NgR activity. In slice cultures of the optic pathway, blocking NgR with a peptide antagonist produced significant reduction in the uncrossed projection but had no effect on the crossing axons. This result was confirmed by treating cultures with an anti-Nogo functional blocking antibody. In vitro coculture assays of retina and optic chiasm showed that NgR was selectively reduced on neurites and growth cones from dorsonasal retina when they contacted chiasm cells, but not on those from ventrotemporal retina. These findings provide evidence that Nogo signaling is involved in directing the growth of axons in the mouse optic chiasm and that this process relies on a differential regulation of NgR on axons from the dorsonasal and ventrotemporal retina.

Distribution of neurotrophin-3 during the ontogeny and regeneration of the lizard (Gallotia galloti) visual system.

We have previously described the spontaneous regeneration of retinal ganglion cell axons after optic nerve (ON) transection in the adult Gallotia galloti. As neurotrophin-3 (NT-3) is involved in neuronal differentiation, survival and synaptic plasticity, we performed a comparative immunohistochemical study of NT-3 during the ontogeny and regeneration (after 0.5, 1, 3, 6, 9, and 12 months postlesion) of the lizard visual system to reveal its distribution and changes during these events. For characterization of NT-3(+) cells, we performed double labelings using the neuronal markers HuC-D, Pax6 and parvalbumin (Parv), the microglial marker tomato lectin or Lycopersicon esculentum agglutinin (LEA), and the astroglial markers vimentin (Vim) and glial fibrillary acidic protein (GFAP). Subpopulations of retinal and tectal neurons were NT-3(+) from early embryonic stages to adulthood. Nerve fibers within the retinal nerve fiber layer, both plexiform layers and the retinorecipient layers in the optic tectum (OT) were also stained. In addition, NT-3(+)/GFAP(+) and NT-3(+)/Vim(+) astrocytes were detected in the ON, chiasm and optic tract in postnatal and adult lizards. At 1 month postlesion, abundant NT-3(+)/GFAP(+) astrocytes and NT-3(-)/LEA(+) microglia/macrophages were stained in the lesioned ON, whereas NT-3 became downregulated in the experimental retina and OT. Interestingly, at 9 and 12 months postlesion, the staining in the experimental retina resembled that in control animals, whereas bundles of putative regrown fibers showed a disorganized staining pattern in the OT. Altogether, we demonstrate that NT-3 is widely distributed in the lizard visual system and its changes after ON transection might be permissive for the successful axonal regrowth.

Molecular regulation of visual system development: more than meets the eye.

Vertebrate eye development has been an excellent model system to investigate basic concepts of developmental biology ranging from mechanisms of tissue induction to the complex patterning and bidimensional orientation of the highly specialized retina. Recent advances have shed light on the interplay between numerous transcriptional networks and growth factors that are involved in the specific stages of retinogenesis, optic nerve formation, and topographic mapping. In this review, we summarize this recent progress on the molecular mechanisms underlying the development of the eye, visual system, and embryonic tumors that arise in the optic system.

Peculiar and typical oligodendrocytes are involved in an uneven myelination pattern during the ontogeny of the lizard visual pathway.

We studied the myelination of the visual pathway during the ontogeny of the lizard Gallotia galloti using immunohistochemical methods to stain the myelin basic protein (MBP) and proteolipid protein (PLP/DM20), and electron microscopy. The staining pattern for the PLP/DM20 and MBP overlapped during the lizard ontogeny and was first observed at E39 in cell bodies and fibers located in the temporal optic nerve, optic chiasm, middle optic tract, and in the stratum album centrale of the optic tectum (OT). The expression of these proteins extended to the nerve fiber layer (NFL) of the temporal retina and to the outer strata of the OT at E40. From hatching onwards, the labeling became stronger and extended to the entire visual pathway. Our ultrastructural data in postnatal and adult animals revealed the presence of both myelinated and unmyelinated retinal ganglion cell axons in all visual areas, with a tendency for the larger axons to show the thicker myelin sheaths. Moreover, two kinds of oligodendrocytes were described: peculiar oligodendrocytes displaying loose myelin sheaths were only observed in the NFL, whereas typical medium electron-dense oligodendrocytes displaying compact myelin sheaths were observed in the rest of the visual areas. The weakest expression of the PLP/DM20 in the NFL of the retina appears to be linked to the loose appearance of its myelin sheaths. We conclude that typical and peculiar oligodendrocytes are involved in an uneven myelination process, which follows a temporo-nasal and rostro-caudal gradient in the retina and ON, and a ventro-dorsal gradient in the OT.

A role for Nr-CAM in the patterning of binocular visual pathways.

Retinal ganglion cell (RGC) axons diverge within the optic chiasm to project to opposite sides of the brain. In mouse, contralateral RGCs are distributed throughout the retina, whereas ipsilateral RGCs are restricted to the ventrotemporal crescent (VTC). While repulsive guidance mechanisms play a major role in the formation of the ipsilateral projection, little is known about the contribution of growth-promoting interactions to the formation of binocular visual projections. Here, we show that the cell adhesion molecule Nr-CAM is expressed by RGCs that project contralaterally and is critical for the guidance of late-born RGCs within the VTC. Blocking Nr-CAM function causes an increase in the size of the ipsilateral projection and reduces neurite outgrowth on chiasm cells in an age- and region-specific manner. Finally, we demonstrate that EphB1/ephrin-B2-mediated repulsion and Nr-CAM-mediated attraction comprise distinct molecular programs that each contributes to the proper formation of binocular visual pathways.

Variations in the architecture and development of the vertebrate optic chiasm.

At the vertebrate optic chiasm there is major change in fibre order and, in many animals, a separation of fibres destined for different hemispheres of the brain. However, the structure of this region is not uniform among all species but rather shows marked variations both in terms of its gross architecture and the pathways taken by different fibres. There also are striking differences in the developmental mechanisms sculpting this region even between closely related animals. In spite of this, recent studies have provided strong evidence for a remarkable degree of conservation in the molecular nature of the guidance signals and regulatory genes driving chiasmatic development. Here differences and similarities in chiasmatic organisation and development between separate groups of animals will be reviewed. While it may not be possible to ascribe a single set of factors that are universal components of the vertebrate chiasm, there are both strikingly similar elements as well as diverse features to the development, organisation and architecture of this region. This review aims to highlight key issues in the organisation and development of the vertebrate optic chiasm with a focus on comparing and contrasting the data that has been gleaned to date from different vertebrate groups.

The optic chiasm as a midline choice point.

The mouse optic chiasm is a model for axon guidance at the midline and for analyzing how binocular vision is patterned. Recent work has identified several molecular players that influence the binary decision that retinal ganglion cells make at the optic chiasm, to either cross or avoid the midline. An ephrin-B localized to the midline, together with an EphB receptor and a zinc-finger transcription factor expressed exclusively in the ventrotemporal retina where ipsilaterally projecting retinal ganglion cells are located, comprise a molecular program for the uncrossed pathway. In addition, the mechanisms for axon divergence in the optic chiasm are discussed in the context of other popular models for midline axon guidance.

Generating X: formation of the optic chiasm.

At the optic chiasm, axons from either eye meet and decide whether to cross contralaterally or turn back ipsilaterally. Here, the guidance ligand Slit and its receptor Robo control not whether axons cross (as in other midline decisions), but where the chiasm forms. Whether axons cross is instead controlled by the transcription factor Zic2 and the guidance receptor EphB1, as shown by two papers in the current issues of Neuron and Cell (Herrera et al. and Williams et al.). Surprisingly, this mechanism is conserved evolutionarily from frogs to mammals.

Changes in expression of fibroblast growth factor receptors during development of the mouse retinofugal pathway.

Retinal axons undergo several changes in organization as they pass through the region of the optic chiasm and optic tract. We used immunocytochemistry to examine the possible involvement of fibroblast growth factor receptors (FGFR) in these changes in retinal axon growth. In the retina, at all ages examined, prominent staining for FGFR was seen in the optic fiber layer and at the optic disk. At embryonic day 15 (E15), FGFR immunoreactivity was also detected in the ganglion cell layer, as defined by immunoreactivity for islet-1. At later developmental stages (E16 to postnatal day 0), FGFR were found in the optic fiber layer and the inner plexiform layer. In the ventral diencephalon, immunostaining for FGFR was first detected at E13 in a group of cells posterior to the chiasm. These cells appeared to match the neurons that are immunopositive for the stage-specific embryonic antigen-1 (SSEA-1). FGFR staining was also found on the retinal axons at E13. At E14-E16, when most axons are growing across the chiasm and the tract, a dynamic pattern of FGFR immunoreactivity was observed on the retinal axons. The staining was reduced when axons reached the midline but was increased when axons reached the threshold of the optic tract. These results suggest that axon growth and fiber patterning in distinct regions of the retinofugal pathway are in part controlled by a regulated expression of FGFR. Furthermore, the axons with elevated FGFR expression in the optic tract have a posterior border of rich FGFR expression in the lateral part of the diencephalon. This region overlaps with a lateral extension of the SSEA-1-positive cells, suggesting a possible relation of these cells to the elevated expression of FGFR.

Architecture of the optic chiasm and the mechanisms that sculpt its development.

At the optic chiasm the two optic nerves fuse, and fibers from each eye cross the midline or turn back and remain uncrossed. Having adopted their pathways the fibers separate to form the two optic tracts. Research into the architecture and development of the chiasm has become an area of increasing interest. Many of its mature features are complex and vary between different animal types. It is probable that numerous factors sculpt its development. The separate ganglion cell classes cross the midline at different locations along the length of the chiasm, reflecting their distinct periods of production as the chiasm develops in a caudo-rostral direction. In some mammals, uncrossed axons are mixed with crossed axons in each hemi-chiasm, whereas in others they remain segregated. These configurations are the product of different developmental mechanisms. The morphology of the chiasm changes significantly during development. Neurons, glia, and the signals they produce play a role in pathway selection. In some animals fiber-fiber interactions are also critical, but only where crossed and uncrossed pathways are mixed in each hemi-chiasm. The importance of the temporal dimension in chiasm development is emphasized by the fact that in some animals uncrossed ganglion cells are generated abnormally early in relation to their retinal location. Furthermore, in albinos, where many cells do not exit the cell cycle at normal times, there are systematic chiasmatic abnormalities in ganglion cell projections.

Ephrin-B regulates the Ipsilateral routing of retinal axons at the optic chiasm.

In Xenopus tadpoles, all retinal ganglion cells (RGCs) send axons contralaterally across the optic chiasm. At metamorphosis, a subpopulation of EphB-expressing RGCs in the ventrotemporal retina begin to project ipsilaterally. However, when these metamorphic RGCs are grafted into embryos, they project contralaterally, suggesting that the embryonic chiasm lacks signals that guide axons ipsilaterally. Ephrin-B is expressed discretely at the chiasm of metamorphic but not premetamorphic Xenopus. When expressed prematurely in the embryonic chiasm, ephrin-B causes precocious ipsilateral projections from the EphB-expressing RGCs. Ephrin-B is also found in the chiasm of mammals, which have ipsilateral projections, but not in the chiasm of fish and birds, which do not. These results suggest that ephrin-B/EphB interactions play a key role in the sorting of axons at the vertebrate chiasm.

Concentration of astrocytic filaments at the retinal optic nerve junction is coincident with the absence of intra-retinal myelination: comparative and developmental evidence.

The structure of the lamina cribrosa (LC) and astrocytic density were examined in various species with and without intra-retinal myelination. Sections of optic nerve from various species were stained with Milligan's trichrome or antibodies to glial fibrillary acidic protein, myelin basic protein (MBP) and antibody O4. Marmoset, flying fox, cat, and sheep, which lack intraretinal myelination, were shown to possess a well-developed LC as well as a marked concentration of astrocytic filaments distal to the LC. Rat and mouse, which lack intraretinal myelination, lacked a well-developed LC but exhibited a marked concentration of astrocytic filaments in this region. Rabbit and chicken, which exhibit intraretinal myelination, lacked both a well-developed LC and a concentration of astrocytes at the retinal optic nerve junction (ROJ). A marked concentration of astrocytes at the ROJ of human fetuses was also apparent at 13 weeks of gestation, prior to myelination of the optic nerve; in contrast, the LC was not fully developed even at birth. This concentration of astrocytes was located distal to O4 and MBP immunoreactivity in human optic nerve, and coincided with the site of initial myelination of ganglion cell axons in marmoset and rat. Myelination proceeded from the chiasm towards the retinal end of the human optic nerve. Moreover, the outer limit of oligodendrocyte precursor cells (OPC) migration into the rabbit retina was restricted by the outer limit of astrocyte spread. These observations indicate that a concentration of astrocytic filaments at the ROJ is coincident with the absence of intraretinal myelination. Differential expression of tenascin-C by astrocytes at the ROJ appears to contribute to the molecular barrier to OPC migration (see Bartsch et al., 1994), while expression of the homedomain protein Vax 1 by glial cells at the optic nerve head appears to inhibit migration of retinal pigment epithelial cells into the optic nerve (see Bertuzzi et al., 1999). These observations combined with our present comparative and developmental data lead us to suggest that the astrocytes at the ROJ serve to regulate cellular traffic into and out of the retina.

Establishment of the FMRFamide-immunoreactive olfacto-retinalis pathway during ontogeny of the rainbow trout (Oncorhynchus mykiss).

Migration of neurons is one of the mechanisms establishing normal central nervous system connectivity during ontogeny. Proper timing of axonal sprouting is relevant in the same context. In the present study, we used the immunoreactivity of the tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2) to visualize the olfacto-retinalis projection during trout ontogeny. It starts to innervate the retina two to four weeks after hatching, in contrast to reports on salmon where it only appears after the fish are imprinted on their natal stream.

Goosecoid-like (Gscl), a candidate gene for velocardiofacial syndrome, is not essential for normal mouse development.

Velocardiofacial syndrome (VCFS) and DiGeorge syndrome (DGS) are characterized by a wide spectrum of abnormalities, including conotruncal heart defects, velopharyngeal insufficiency, craniofacial anomalies and learning disabilities. In addition, numerous other clinical features have been described, including frequent psychiatric illness. Hemizygosity for a 1.5-3 Mb region of chromosome 22q11 has been detected in >80% of VCFS/DGS patients. It is thought that a developmental field defect is responsible for many of the abnormalities seen in these patients and that the defect occurs due to reduced levels of a gene product active in early embryonic development. Goosecoid-like ( GSCL ) is a homeobox gene which is present in the VCFS/DGS commonly deleted region. The mouse homolog, Gscl, is expressed in mouse embryos as early as E8.5. Gscl is related to Goosecoid ( Gsc ), a gene required for proper craniofacial development in mice. GSCL has been considered an excellent candidate for contributing to the developmental defects in VCFS/DGS patients. To investigate the role of Goosecoid-like in VCFS/DGS etiology, we disrupted the Gscl gene in mouse embryonic stem cells and produced mice that transmit the disrupted allele. Mice that are homozygous for the disrupted allele appear to be normal and they do not exhibit any of the anatomical abnormalities seen in VCFS/DGS patients. RNA in situ hybridization to mouse embryo sections revealed that Gscl is expressed at E8.5 in the rostral region of the foregut and at E11.5 and E12.5 in the developing brain, in the pons region and in the choroid plexus of the fourth ventricle. Although the gene inactivation experiments indicate that haploinsufficiency for GSCL is unlikely to be the sole cause of the developmental field defect thought to be responsible for many of the abnormalities in VCFS/DGS patients, its localized expression during development could suggest that hemizygosity for GSCL, in combination with hemizygosity for other genes in 22q11, contributes to some of the developmental defects as well as the behavioral anomalies seen in these patients. The mice generated in this study should help in evaluating these possibilities.