J. Martin Collinson

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity—the absence of blood vessels in the cornea—is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

Clonal analysis of Patterns of growth, stem cell activity, and cell movement during the development and maintenance of the murine corneal epithelium

Patterns of growth and cell movement in the developing and adult corneal epithelium were investigated by analysing clonal patches of LacZ‐expressing cells in chimeric and X‐inactivation mosaic mice. It was found that cell proliferation throughout the basal corneal epithelium during embryogenesis and early postnatal life creates a disordered mosaic pattern of LacZ+ clones that contrasts with patterns of proliferation and striping produced during the later embryonic stages of retinal pigmented epithelium development. The early mosaic pattern in the corneal epithelium is replaced in the first 12 postnatal weeks by an ordered pattern of radial stripes or sectors that reflects migration without mixing of the progeny of clones of limbal stem cells. In contrast to previous assumptions, it was found that maturation of the activity of limbal stem cells and the pattern of migration of their progeny are delayed for several weeks postnatally. No evidence was found for immigration of the progeny of stem cells until the 5th postnatal week. There are approximately 100 clones of limbal stem cells initially, and clones are lost during postnatal life. Our studies provide a new assay for limbal and corneal defects in mutant mice.

The roles of Pax6 in the cornea, retina, and olfactory epithelium of the developing mouse embryo

The roles of Pax6 were investigated in the murine eye and the olfactory epithelium by analysing gene expression and distribution of Pax6(-/-) cells in Pax6(+/+) <--> Pax6(-/-) chimeras. It was found that between embryonic days E10.5 and E16.5 Pax6 is autonomously required for cells to contribute fully not only to the corneal epithelium, where Pax6 is expressed at high levels, but also to the to the corneal stroma and endothelium, where the protein is detected at very low levels. Pax6(-/-) cells contributed only poorly to the neural retina, forming small clumps of cells that were normally restricted to the ganglion cell layer at E16.5. Pax6(-/-) cells in the retinal pigment epithelium could express Trp2, a component of the pigmentation pathway, at E14.5 and a small number went on to differentiate and produce pigment at E16.5. The segregation and near-exclusion of mutant cells from the nasal epithelium mirrored the behaviour of mutant cells in other developmental contexts, particularly the lens, suggesting that common primary defects may be responsible for diverse Pax6-related phenotypes.

Embryonic Submandibular Gland Morphogenesis: Stage-Specific Protein Localization of FGFs, BMPs, Pax6 and Pax9 in Normal Mice and Abnormal SMG Phenotypes in FgfR2-IIIc+/Δ, BMP7–/– and Pax6–/–Mice

Embryonic submandibular salivary gland (SMG) initiation and branching morphogenesis are dependent on cell-cell communications between and within epithelium and mesenchyme. Such communications are typically mediated in other organs (teeth, lung, lacrimal glands) by growth factors in such a way as to translate autocrine, juxtacrine and paracrine signals into specific gene responses regulating cell division and histodifferentiation. Using Wnt1-Cre/R26R transgenic mice, we demonstrate that embryonic SMG mesenchyme is derived exclusively from cranial neural crest. This origin contrasts to that known for tooth mesenchyme, previously shown to be derived from both neural crest and nonneural crest cells. Thus, although both SMGs and teeth are mandibular derivatives, we can expect overlap and differences in the details of their early inductive interactions. In addition, since embryonic SMG branching morphogenesis is analogous to that seen in other branching organs, we also expect similarities of expression regarding those molecules known to be ubiquitous regulators of morphogenesis. In this study, we performed an analysis of the distribution of specific fibroblast growth factors (FGFs), FGF receptors, bone morphogenetic proteins (BMPs) and Pax transcription factors, previously shown to be important for tooth development and/or branching morphogenesis, from the time of initiation of embryonic SMG development until early branching morphogenesis. In addition, we report abnormal SMG phenotypes in FgfR2- IIIc+/Δ, BMP7–/–and Pax6–/– mice. Our results, in comparison with functional studies in other systems, suggest that FGF-2/FGFR-1, FGF-8/FGFR-2(IIIc) and FGF-10/FGFR-2(IIIb) signaling have different paracrine and juxtacrine functions during SMG initial bud formation and branching. Finally, our observations of abnormal SMGs in BMP7–/– and Pax6–/–indicate that both BMP7 and Pax6 play important roles during embryonic SMG branching morphogenesis.

Primary defects in the lens underlie complex anterior segment abnormalities of the Pax6 heterozygous eye

We describe lens defects in heterozygous small eye mice, and autonomous deficiencies of Pax6+/− cells in the developing lens of Pax6+/+ ↔ Pax6+/− chimeras. Two separate defects of the lens were identified by analyzing the distribution of heterozygous cells in chimeras: Pax6+/− cells are less readily incorporated into the lens placode than wild type, and those that are incorporated into the lens are not maintained efficiently in the proliferating lens epithelium. The lens of chimeric eyes is, therefore, predominantly wild type from embryonic day 16.5 onwards, whereas heterozygous cells contribute normally to all other eye tissues. Eye size and defects of the iris and cornea are corrected in fetal and adult chimeras with up to 80% mutant cells. Therefore, these aspects of the phenotype may be secondary consequences of primary defects in the lens, which has clinical relevance for the human aniridia (PAX6+/−) phenotype.

Pax6 regulates regional development and neuronal migration in the cerebral cortex.

Mutations in the Pax6 gene disrupt telencephalic development, resulting in a thin cortical plate, expansion of proliferative layers, and the absence of the olfactory bulb. The primary defect in the neuronal cell population of the developing cerebral cortex was analysed by using mouse chimeras containing a mixture of wild-type and Pax6-deficient cells. The chimeric analysis shows that Pax6 influences cellular activity throughout corticogenesis. At early stages, Pax6-deficient and wildtype cells segregate into exclusive patches, indicating an inability of different cell genotypes to interact. At later stages, cells are sorted further based on telencephalic domains. Pax6-deficient cells are specifically reduced in the mediocaudal domain of the dorsal telencephalon, indicating a role in regionalization. In addition, Pax6 regulates the process of radial migration of neuronal precursors. Loss of Pax6 particularly affects movement of neuronal precursors at the subventricular zone/intermediate zone boundary at a transitional migratory phase essential for entry into the intermediate zone. We suggest that the primary role of Pax6 is the continual regulation of cell surface properties responsible for both cellular identity and radial migration, defects of which cause regional cell sorting and abnormalities of migration in chimeras.

The roles of calcium signaling and ERK1/2 phosphorylation in a Pax6+/- mouse model of epithelial wound-healing delay

BackgroundCongenital aniridia caused by heterozygousity at the PAX6 locus is associated with ocular surface disease including keratopathy. It is not clear whether the keratopathy is a direct result of reduced PAX6 gene dosage in the cornea itself, or due to recurrent corneal trauma secondary to defects such as dry eye caused by loss of PAX6 in other tissues. We investigated the hypothesis that reducing Pax6 gene dosage leads to corneal wound-healing defects. and assayed the immediate molecular responses to wounding in wild-type and mutant corneal epithelial cells.ResultsPax6+/- mouse corneal epithelia exhibited a 2-hour delay in their response to wounding, but subsequently the cells migrated normally to repair the wound. Both Pax6+/+ and Pax6+/- epithelia activated immediate wound-induced waves of intracellular calcium signaling. However, the intensity and speed of propagation of the calcium wave, mediated by release from intracellular stores, was reduced in Pax6+/- cells. Initiation and propagation of the calcium wave could be largely decoupled, and both phases of the calcium wave responses were required for wound healing. Wounded cells phosphorylated the extracellular signal-related kinases 1/2 (phospho-ERK1/2). ERK1/2 activation was shown to be required for rapid initiation of wound healing, but had only a minor effect on the rate of cell migration in a healing epithelial sheet. Addition of exogenous epidermal growth factor (EGF) to wounded Pax6+/- cells restored the calcium wave, increased ERK1/2 activation and restored the immediate healing response to wild-type levels.ConclusionThe study links Pax6 deficiency to a previously overlooked wound-healing delay. It demonstrates that defective calcium signaling in Pax6+/- cells underlies this delay, and shows that it can be pharmacologically corrected. ERK1/2 phosphorylation is required for the rapid initiation of wound healing. A model is presented whereby minor abrasions, which are quickly healed in normal corneas, transiently persist in aniridic patients, compromising the corneal stroma.

Transient expression of neurofascin by oligodendrocytes at the onset of myelinogenesis : Implications for mechanisms of axon-glial interaction

Cell adhesion molecules (CAMs) must play a crucial role in both the initiation and signalling of axon‐glial contact. However, the proteins that permit myelinating oligodendrocytes to recognize the axons that they ensheath in the developing CNS are unknown. By a subtractive cDNA library strategy, we have identified neurofascin as a powerful candidate for such a molecule. Neurofascin is strongly but transiently up‐regulated in oligodendrocytes at the onset of myelinogenesis. Once oligodendrocytes have engaged their target axons the protein plays no further part, since the expression of the gene declines precipitously, in contrast to that of the major myelin component proteolipid protein, which remains elevated. After the initial surge of neurofascin expression in oligodendrocytes, there is a shift to a predominantly neuronal localization that persists into adulthood. Hence neurofascin in oligodendrocytes is unlikely to serve a function in the stabilization of the multilamellar sheath around the axon. The major neurofascin isoform of oligodendrocytes contains the third fibronectin type 3 (FNIII) repeat but lacks the mucin‐like domain which supports the view that neurofascin isoforms are differentially expressed in the nervous system. Among the genes that are up‐regulated during the terminal differentiation of the oligodendrocyte, neurofascin is unique in displaying a transient pattern of expression at the early stages of myelination. We propose that this CAM not only has a role in mediating axon recognition but also signals axonal contact through its links with the actin cytoskeleton. GLIA 23:11–23, 1998.

Control of Patterns of Corneal Innervation by Pax6

PURPOSE Corneal nerves play essential roles in maintaining the ocular surface through provision of neurotrophic support, but genetic control of corneal innervation is poorly understood. The possibility of a neurotrophic failure in ocular surface disease associated with heterozygosity at the Pax6 locus (aniridia-related keratopathy [ARK]) was investigated. METHODS Patterns of corneal innervation were studied during development and aging in mice with different Pax6 dosages and in chimeras. Immunohistochemistry and ELISA-based assays were used to determine the molecular basis of defects seen in Pax6 mutants, and wound healing assays were performed. RESULTS In adults, the Pax6(+/-) epithelium was less densely innervated than the wild-type epithelium, and radial projection of epithelial nerves was disrupted. Neurotrophic support of the corneal epithelium appeared normal. Directed nerve projection correlated with patterns of epithelial cell migration in adult wild-types, but innervation defects observed in Pax6(+/-) mice were not fully corrected in wound healing or chimeric models where directed epithelial migration was restored. CONCLUSIONS Pax6 dosage nonautonomously controls robust directed radial projection of corneal neurons, and the guidance cues for growth cone guidance are not solely dependent on directed epithelial migration. There is little evidence that ARK represents neurotrophic keratitis.

PAX6 dosage effects on corneal development, growth, and wound healing.

The requirement for correct dosage of the transcription factor Pax6 during corneal growth and development was investigated using the Pax6‐overexpressing (PAX77) transgenic mouse. Transgenics had a microcornea phenotype due to failure of postnatal growth, associated with reduction in the number of cells layers in the corneal epithelium. Cell cycle progression was monitored using bromodeoxyuridine, p63, cyclin E, and phosphohistone‐3 labeling: proliferation rates were higher in PAX77+ than wild‐type, without a concomitant increase in apoptosis. Hence, failure of proliferation did not underlie microcornea. PAX77+ corneal epithelia had reduced levels of cytokeratin‐12, and exhibited severe wound healing delay that, in contrast to Pax6+/− mice, could not be modulated by exogenous growth factors. PAX77+ lenses showed partial failure of lens fiber differentiation. The data demonstrate that anterior eye development is very sensitive to Pax6 dosage. Although there are similarities between the eye phenotype of Pax6 heterozygotes and overexpressing mice, there are also striking differences. Developmental Dynamics 237:1295‐1306, 2008.