Pax6-dependence of Six3, Eya1 and Dach1 expression during lens and nasal placode induction

The Drosophila eyeless gene plays a central role in fly eye development and controls a subordinate regulatory network consisting of the so, eya and dac genes. All three genes have highly conserved mammalian homologs, suggesting possible conservation of this eye forming regulatory network. sine oculis (so) belongs to the so/Six gene family, and Six3 is prominently expressed in the developing mammalian eye. Eya1 and Dach1 are mammalian homologs of eya and dac, respectively, and although neither Eya1 nor Dach1 knockout mice express prenatal eye defects, possibilities exist for postnatal ocular phenotypes or for functional redundancy between related family members. To examine whether expression relationships analogous to those between ey, so, eya and dac exist in early mammalian oculogenesis, we investigated Pax6, Six3, Eya1 and Dach1 protein expression in murine lens and nasal placode development. Six3 expression in the pre-placode lens ectoderm is initially Pax6-independent, but subsequently both its expression and nuclear localization become Pax6-dependent. Six3, Dach1 and Eya1 nasal expression in pre-placode ectoderm are also initially Pax6-independent, but thereafter become Pax6-dependent. Pax6, Six3, Dach1 and Eya1 are all co-expressed in the developing ciliary marginal zone, a source of retinal stem cells in some vertebrates. An in vitro protein-protein interaction is detected between Six3 and Eya1. Collectively, these findings suggest that the Pax-Eya-Six-Dach network is at best only partly conserved during lens and nasal placode development. However, the findings do not rule out the possibility that such a regulatory network acts at later stages of oculogenesis.     

Step-wise specification of retinal stem cells during normal embryogenesis

The specification of embryonic cells to produce the retina begins at early embryonic stages as a multi-step process that gradually restricts fate potentials. First, a subset of embryonic cells becomes competent to form retina by their lack of expression of endo-mesoderm-specifying genes. From these cells, a more restricted subset is biased to form retina by virtue of their close proximity to sources of bone morphogenetic protein antagonists during neural induction. During gastrulation, the definitive RSCs (retinal stem cells) are specified as the eye field by interactions with underlying mesoderm and the expression of a network of retina-specifying genes. As the eye field is transformed into the optic vesicle and optic cup, a heterogeneous population of RPCs (retinal progenitor cells) forms to give rise to the different domains of the retina: the optic stalk, retinal pigmented epithelium and neural retina. Further diversity of RPCs appears to occur under the influences of cell-cell interactions, cytokines and combinations of regulatory genes, leading to the differentiation of a multitude of different retinal cell types. This review examines what is known about each sequential step in retinal specification during normal vertebrate development, and how that knowledge will be important to understand how RSCs might be manipulated for regenerative therapies to treat retinal diseases.     

3T3 cell lines stably expressing Pax6 or Pax6(5a)--a new tool used for identification of common and isoform specific target genes

Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.     

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye^1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment⁴; however the cells are still capable of forming the different cell types found within the neural retina^1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury⁵, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent^6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation^9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.     

Pax genes in development and maturation of the vertebrate visual system: implications for optic nerve regeneration

Pax genes play a pivotal role in development of the vertebrate visual system. Pax6 is the master control gene for eye development: ectopic expression of Pax6 in Xenopus laevis and Drosphila melanogaster leads to the formation of differentiated eyes on the legs or wings. Pax6 is involved in formation of ganglion cells of the retina, as well as cells of the lens, iris and cornea. In addition Pax6 may play a role in axon guidance in the visual system. Pax2 regulates differentiation of the optic disk through which retinal ganglion cell axons exit the eye. Furthermore, Pax2 plays a critical role in development of the optic chiasm and in the guidance of axons along the contralateral or ipsilateral tracts of the optic nerve to visual targets in the brain. During development Pax7 is expressed in neuronal cells of one of the major visual targets in the brain, the optic tectum/superior colliculus. Neurons expressing Pax7 migrate towards the pia and concentrate in the stratum griseum superficiale (SGFS), the target site for retinal axons. Together, expression of Pax2, 6 and 7 may guide axons during formation of functional retinotectal/collicular projections. Highly regulated Pax gene expression is also observed in mature animals. Moreover, evidence suggests that Pax genes are important for regeneration of the visual system. We are currently investigating Pax gene expression in species that display a range of outcomes of optic nerve regeneration. We predict that such information will provide valuable insights for the induction of successful regeneration of the optic nerve and of other regions of the central nervous system in mammals including man.     

Analysis of the Expression Pattern of Regulatory Genes Pax6, Prox1, and Six3 during Regeneration of Eye Structures in the Newt

We studied tissue-specific expression of homeobox genes Pax6, Prox1, and Six3 during regeneration of the retina and lens. In the native retina, mRNA of Pax6, Prox1, and Six3 was predominantly localized in ganglion cells and in the inner nuclear layer of the retina. Active Pax6, Prox1, and Six3 expression was detected at early stages of regeneration in all proliferating neuroblasts forming the retinal primordium. Low levels of Pax6, Prox1, and Six3 mRNA were revealed in depigmented cells of the pigment epithelium as compared to the proliferating neuroblasts. At the intermediate stage of retinal regeneration, the distribution of Pax6, Prox1, and Six3 mRNA was diffuse and even all over the primordium. During differentiation of the cellular layers in the course of retinal regeneration, Pax6, Prox1, and Six3 mRNA was predominantly localized in ganglion cells and in the inner part of the inner nuclear layer, which was similar to the native retina. An increased expression was revealed in the peripheral regenerated retina where multipotent cells were localized. The dual role of regulatory genes Pax6, Prox1, and Six3 during regeneration of eye structures has been revealed; these genes controlled cell proliferation and subsequent differentiation of ganglion, amacrine, and horizontal cells. High hybridization signal of all studied genes was revealed in actively proliferating epithelial cells of the native and regenerating lens, while the corneal epithelium demonstrated a lower signal. Pax6 and Prox1 expression was also revealed in single choroid cells of the regenerating eye.     

Function of Rx, but not Pax6, is essential for the formation of retinal progenitor cells in mice

Rx plays a critical role in eye formation. Targeted elimination of Rx results in embryos that do not develop eyes. In this study, we have investigated the expression of Otx2, Six3, and Pax6 in Rx deficient embryos. We find that these genes show normal activation in the anterior neural plate in Rx-/- embryos, but they are not upregulated in the area of the neural plate that would form the primordium of the optic vesicle. In contrast, in homozygous Small eye embryos that lack Pax6 function, Rx shows normal activation in the anterior neural plate and normal upregulation in the optic vesicle/retinal progenitor cells. This suggests that neither Rx expression nor the formation of retinal progenitor cells is dependent on a functional copy of the Pax6 gene, but that Pax6 expression and the formation of the progenitor cells of the optic cup is dependent on a functional copy of the Rx gene.     

Alterations of rx1 and pax6 expression levels at neural plate stages differentially affect the production of retinal cell types and maintenance of retinal stem cell qualities

rx1 and pax6 are necessary for the establishment of the vertebrate eye field and for the maintenance of the retinal stem cells that give rise to multiple retinal cell types. They also are differentially expressed in cellular layers in the retina when cell fates are being specified, and their expression levels differentially affect the production of amacrine cell subtypes. To determine whether rx1 and pax6 expression after the eye field is established simply maintains stem cell-like qualities or affects cell type differentiation, we used hormone-inducible constructs to increase or decrease levels/activity of each protein at two different neural plate stages. Our results indicate that rx1 regulates the size of the retinal stem cell pool because it broadly affected all cell types, whereas pax6 regulates more restricted retinal progenitor cells because it selectively affected different cell types in a time-dependent manner. Analysis of rx1 and pax6 effects on proliferation, and expression of stem cell or differentiation markers demonstrates that rx1 maintains cells in a stem cell state by promoting proliferation and delaying expression of neural identity and differentiation markers. Although pax6 also promotes proliferation, it differentially regulates neural identity and differentiation genes. Thus, these two genes work in parallel to regulate different, but overlapping aspects of retinal cell fate determination.     

Dominant inhibition of lens placode formation in mice

The lens in the vertebrate eye has been shown to be critical for proper differentiation of the surrounding ocular tissues including the cornea, iris and ciliary body. In mice, previous investigators have assayed the consequences of molecular ablation of the lens. However, in these studies, lens ablation was initiated (and completed) after the cornea, retina, iris and ciliary body had initiated their differentiation programs thereby precluding analysis of the early role of the lens in fate determination of these tissues. In the present study, we have ablated the lens precursor cells of the surface ectoderm by generation of transgenic mice that express an attenuated version of diphtheria toxin (Tox176) linked to a modified Pax6 promoter that is active in the lens ectodermal precursors. In these mice, lens precursor cells fail to express Sox2, Prox1 and αA-crystallin and die before the formation of a lens placode. The Tox176 mice also showed profound alterations in the corneal differentiation program. The corneal epithelium displayed histological features of the skin, and expressed markers of skin differentiation such as Keratin 1 and 10 instead of Keratin 12, a marker of corneal epithelial differentiation. In the Tox176 mice, in the absence of the lens, extensive folding of the retina was seen. However, differentiation of the major cell types in the retina including the ganglion, amacrine, bipolar and horizontal cells was not affected. Unexpectedly, ectopic placement of the retinal pigmented epithelium was seen between the folds of the retina. Initial specification of the presumptive ciliary body and iris at the anterior margins of the retina was not altered in the Tox176 mice but their subsequent differentiation was blocked. Lacrimal and Harderian glands, which are derived from the Pax6-expressing surface ectodermal precursors, also failed to differentiate. These results suggest that, in mice, specification of the retina, ciliary body and iris occurs at the very outset of eye development and independent of the lens. In addition, our results also suggest that the lens cells of the surface ectoderm may be critical for the proper differentiation of the corneal epithelium.     

Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. These results indicate that Pax6 may have different roles during the different phases of RPE to retinal TD, acting as an early retinal determinant and later directing progenitor cell fate.