VEGF-A and neuropilin 1 (NRP1) shape axon projections in the developing CNS via dual roles in neurons and blood vessels.

Visual information is relayed from the eye to the brain via retinal ganglion cell (RGC) axons. Mice lacking NRP1 or NRP1-binding VEGF-A isoforms have defective RGC axon organisation alongside brain vascular defects. It is not known whether axonal defects are caused exclusively by defective VEGF-A signalling in RGCs or are exacerbated by abnormal vascular morphology. Targeted NRP1 ablation in RGCs with a Brn3bCre knock-in allele reduced axonal midline crossing at the optic chiasm and optic tract fasciculation. In contrast, Tie2-Cre-mediated endothelial NRP1 ablation induced axon exclusion zones in the optic tracts without impairing axon crossing. Similar defects were observed in Vegfa120/120 and Vegfa188/188 mice, which have vascular defects as a result of their expression of single VEGF-A isoforms. Ectopic midline vascularisation in endothelial Nrp1 and Vegfa188/188 mutants caused additional axonal exclusion zones within the chiasm. As in vitro and in vivo assays demonstrated that vessels do not repel axons, abnormally large or ectopically positioned vessels are likely to present physical obstacles to axon growth. We conclude that proper axonal wiring during brain development depends on the precise molecular control of neurovascular co-patterning.

DSCAM promotes axon fasciculation and growth in the developing optic pathway.

Although many aspects of optic pathway development are beginning to be understood, the mechanisms promoting the growth of retinal ganglion cell (RGC) axons toward visual targets remain largely unknown. Down syndrome cell adhesion molecule (Dscam) is expressed by mouse RGCs shortly after they differentiate at embryonic day 12 and is essential for multiple aspects of postnatal visual system development. Here we show that Dscam is also required during embryonic development for the fasciculation and growth of RGC axons. Dscam is expressed along the developing optic pathway in a pattern consistent with a role in regulating RGC axon outgrowth. In mice carrying spontaneous mutations in Dscam (Dscamdel17 ; Dscam2J), RGC axons pathfind normally, but growth from the chiasm toward their targets is impaired, resulting in a delay in RGC axons reaching the dorsal thalamus compared with that seen in wild-type littermates. Conversely, Dscam gain of function results in exuberant growth into the dorsal thalamus. The growth of ipsilaterally projecting axons is particularly affected. Axon organization in the optic chiasm and tract and RGC growth cone morphologies are also altered in Dscam mutants. In vitro DSCAM promotes RGC axon growth and fasciculation, and can act independently of cell contact. In vitro and in situ DSCAM is required both in the RGC axons and in their environment for the promotion of axon outgrowth, consistent with a homotypic mode of action. These findings identify DSCAM as a permissive signal that promotes the growth and fasciculation of RGC axons, controlling the timing of when RGC axons reach their targets.

Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium.

PURPOSE: To investigate the roles of intracellular signaling elicited by Hedgehog (Hh) ligands in corneal maintenance and wound healing. METHODS: The expression of Hedgehog pathway components in the cornea was assayed by immunohistochemistry, western blot and reverse-transcription polymerase chain reaction (RT-PCR), in wild-type mice and mice that were heterozygous null for the gene encoding the transcription factor, paired box gene 6 (Pax6).  Corneal epithelial wound healing and cell migration assays were performed after pharmacological upregulation and downregulation of the hedgehog pathway.  Reporter mice, mosaic for expression of the gene encoding ß-galactosidase (LacZ), were crossed to Pax6(+/-) mice, mice heterozygous for the gene encoding GLI-Kruppel family member GLI3, and Pax6(+/-)Gli3(+/-) double heterozygotes, to assay patterns of cell migration and corneal epithelial organization in vivo. RESULTS: Corneal epithelial wound healing rates increased in response to application of Sonic hedgehog (Shh), but only in mice with wild-type Pax6 dosage.  Downregulation of Hedgehog signalling inhibited corneal epithelial cell proliferation.  Pax6(+/-) corneal epithelia showed increased proliferation in response to exogenous Shh, but not increased migration. Desert hedgehog (Dhh) was shown to be the major endogenous ligand, with Shh detectable only by RT-PCR and only after epithelial wounding. The activity of phosphatidylinositol-3-OH kinase-γ (PI3Kγ) was not required for the increased migration response in response to Shh.  Nuclear expression of the activator form of the transcription factor Gli3 (which mediates Hh signalling) was reduced in Pax6(+/-) corneal epithelia. Pax6(+/-)Gli3(+/-) double heterozygotes showed highly disrupted patterns of clonal arrangement of cells in the corneal epithelium. CONCLUSIONS: The data show key roles for endogenous Dhh signalling in maintenance and regeneration of the corneal epithelium, demonstrate an interaction between Pax6 and Hh signalling in the corneal epithelium, and show that failure of Hh signalling pathways is a feature of Pax6(+/-) corneal disease that cannot be remedied pharmacologically by addition of the ligands.

Cell autonomy of DSCAM function in retinal development.

Cell adhesion molecules (CAMs) provide identifying cues by which neural architecture is sculpted. The Down Syndrome Cell Adhesion Molecule (DSCAM) is required for many neurodevelopmental processes in different species and also has several potential mechanisms of activity, including homophilic adhesion, homophilic repulsion and heterophilic interactions. In the mouse retina, Dscam is expressed in many, but not all neuronal subtypes. Mutations in Dscam cause the fasciculation of dendrites of neighboring homotypic neurons, indicating a role in self-avoidance among cells of a given type, a disruption of the non-random patterning of their cell bodies, and a decrease in developmental cell death in affected cell populations. In order to address how DSCAM facilitates retinal pattering, we developed a conditional allele of Dscam to use alongside existing Dscam mutant mouse strains. Conditional deletion of Dscam reproduces cell spacing, cell number and dendrite arborization defects. Inducible deletion of Dscam and retinal ganglion cell depletion in Brn3b mutant retinas both indicate that these DSCAM-mediated phenotypes can occur independently. In chimeric retinas, in which wild type and Dscam mutant cells are comingled, Dscam mutant cells entangle adjacent wild type cells of the same type, as if both cells were lacking Dscam, consistent with DSCAM-dependent cell spacing and neurite arborization being mediated through homophilic binding cell-to-cell. Deletion of Dscam in specific cell types causes cell-type-autonomous cell body spacing defects, indicating that DSCAM mediates arborization and spacing by acting within given cell types. We also examine the cell autonomy of DSCAM in laminar stratification and find that laminar disorganization can be caused in a non-cell autonomous fashion. Finally, we find Dscam dosage-dependent defects in developmental cell death and amacrine cell spacing, relevant to the increased cell death and other disorders observed in Down syndrome mouse models and human patients, in which Dscam is present in three copies.

DSCAM and DSCAML1 function in self-avoidance in multiple cell types in the developing mouse retina.

DSCAM and DSCAM-LIKE1 (DSCAML1) serve diverse neurodevelopmental functions, including axon guidance, synaptic adhesion, and self-avoidance, depending on the species, cell type, and gene family member studied. We examined the function of DSCAM and DSCAML1 in the developing mouse retina. In addition to a subset of amacrine cells, Dscam was expressed in most retinal ganglion cells (RGCs). RGCs had fasciculated dendrites and clumped cell bodies in Dscam(-/-) mice, suggesting a role in self-avoidance. Dscaml1 was expressed in the rod circuit, and mice lacking Dscaml1 had fasciculated rod bipolar cell dendrites and clumped AII amacrine cell bodies, also indicating a role in self-avoidance. Neurons in Dscam or Dscaml1 mutant retinas stratified their processes appropriately in synaptic laminae in the inner plexiform layer, and functional synapses formed in the rod circuit in mice lacking Dscaml1. Therefore, DSCAM and DSCAML1 function similarly in self-avoidance, and are not essential for synaptic specificity in the mouse retina.