Expression of Regulatory Homeobox Genes during Retina Regeneration in Adult Newts

We studied molecular-genetic mechanisms of retina regeneration in amphibians and, specifically, expression of the homeobox genes Pax6, Prox1, and Six3 in normal development and during retina regeneration in the newt. Based on the structural analysis of genes in closely related amphibian species, primers were constructed that flank certain regions of these genes. PCR fragments of calculated length were obtained. The relationship of PCR products to the above genes was confirmed by sequencing. A comparative PCR analysis of expression of Pax6, Prox1, and Six3 was carried out in the native and regenerating newt retina, which allowed estimation of the level of expression. cDNA libraries obtained from the native and regenerating retina were used as templates. The libraries were preliminary standardized according to glyceraldehydes-3-phosphate dehydrogenase, an enzyme of general cell metabolism. The genes we studied were expressed in both native and regenerating retina. The level of Pax6 and Prox1 expression increased during regeneration, while that of Six3 decreased. The decrease in the level of Six3 expression could be due to antagonistic interrelations of Prox1 and Six3. The changed level of Prox1 and Six3 expression is a new fact and requires further studies. The interactions between these and other regulatory genes and localization of their expression in the cells of native and regenerating retina will be studied using in situ hybridization and immunohistochemistry.     

Expression of regulatory genes Px6, Otx2, Six3, and FGF2 during newt retina regeneration

Molecular-genetic mechanisms of regeneration of adult newt (Pleurodeles waltl) retina were studied. For the first time, a comparative analysis of the expression of regulatory genes Pax6, Otx2, and Six3 and FGF2 genes encoding signal molecules was performed in the normal retinal pigment epithelium (RPE) and retina and at successive stages of retina regeneration. Cell differentiation types were determined using genetic markers of cell differentiation in the RPE (RPE65) and the retina (βII-tubulin and Rho). Activation of the expression of neurospecific genes Pax6 and Six3 and the growth factor gene FGF2 and suppression of activation of the regulatory gene Otx2 and the RPE65 were observed at the stage of multipotent neuroblast formation in the regenerating retina. The expression of genes Pax6, Six3, and Fgf2 was retained at a later stage of retina regeneration at which the expression of retinal differentiation markers, the genes encoding β II-tubulin (βII-tubulin) and rhodopsin (Rho), was also detected. We assume that the above regulatory genes are multifunctional and control not only transdifferentiation of RPE cells (the key stage of retina regeneration) but also differentiation of regenerating retina cells. The results of this study, demonstrating coexpression of Pax6, Six3, Fgf2, βII-tubulin, and Rho genes, provide indirect evidence for the interaction of regulatory and specific genes during retina regeneration.     

Antibodies against pax6 immunostain amacrine and ganglion cells and neuronal progenitors,but not rod precursors,in the normal and regenerating retina of the goldfish

Pax6 is a developmental regulatory gene that plays a key role in the development of the embryonic brain, eye, and retina. This gene is also expressed in discrete groups of neurons within the adult brain. In this study, antibodies raised against a fusion protein from a zebra fish pax6 cDNA were used to investigate the expression of the pax6 gene in the mature, growing, and regenerating retina of the goldfish. On western blots of retinal proteins, the pax6 antibodies recognize a single band at the approximate size of the zebra fish pax6 protein. In retinal sections, the antibodies label the nuclei of mature amacrine and some ganglion cells. At the retinal margin, where neurogenesis and cellular differentiation continually occur in goldfish, the antibodies label neuronal progenitors and the newly postmitotic neurons. Following injury and during neuronal regeneration, the antibodies label mitotically active progenitors of regenerating neurons. Rod precursors, proliferating cells that normally give rise solely to rod photoreceptors and are the presumed antecedents of the injury-stimulated neuronal progenitors, are not immunostained by antibodies to the pax6 protein. The results of this study document the identity of pax6-expressing cells in the mature retina and demonstrate that in the goldfish pax6 is expressed in neuronal progenitors during both retinal growth and regeneration. © 1996 John Wiley & Sons, Inc.     

Pax6- and Six3-Mediated Induction of Lens Cell Fate in Mouse and Human ES Cells

Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.     

Math3 and NeuroD regulate amacrine cell fate specification in the retina

The basic helix-loop-helix genes Math3 and NeuroD are expressed by differentiating amacrine cells, retinal interneurons. Previous studies have demonstrated that a normal number of amacrine cells is generated in mice lacking either Math3 or NEUROD: We have found that, in Math3-NeuroD double-mutant retina, amacrine cells are completely missing, while ganglion and Müller glial cells are increased in number. In the double-mutant retina, the cells that would normally differentiate into amacrine cells did not die but adopted the ganglion and glial cell fates. Misexpression studies using the developing retinal explant cultures showed that, although Math3 and NeuroD alone only promoted rod genesis, they significantly increased the population of amacrine cells when the homeobox gene Pax6 or Six3 was co-expressed. These results indicate that Math3 and NeuroD are essential, but not sufficient, for amacrine cell genesis, and that co-expression of the basic helix-loop-helix and homeobox genes is required for specification of the correct neuronal subtype.     

Cues intrinsic to the retina induce nAchR gene expression during development

Recent studies of optic nerve regeneration in goldfish have indicated that the optic tectum plays an important role in modulating the induction of nicotinic acetylcholine receptor (nAChR) gene expression in regenerating retinal ganglion cells (Hieber, Agranoff, and Goldman, 1992, J. Neurochem. 58:1009–1015). These observations suggest that induction of these genes is regulated by brain target regions. The appearance of nAChR mRNA in the developing rat retina coincides with a time when ganglion cells are sending axons to their brain targets (Hoover and Goldman, 1992, Exp. Eye Res. 54:561–571). Might a mechanism similar to that seen during goldfish optic nerve regenerationalso mediate induction of nAChR gene expression during development of the mammalian retina? This possibility was tested by either transplanting embryonic rat retina to different brain regions, or explanting it to organ culture and assaying for nAChR gene expression. These studies showed that induction of the nAChR genes in developing rat retina is independent of the environment in which the retina develops. These results indicate that either the retinal microenvironment or a signal intrinsic to the retinal ganglion cell is responsible for this induction. © 1993 John Wiley & Sons, Inc.     

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.     

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.     

Canal Cristae Growth and Fiber Extension to the Outer Hair Cells of the Mouse Ear Require Prox1 Activity

Background

The homeobox gene Prox1 is required for lens, retina, pancreas, liver, and lymphatic vasculature development and is expressed in inner ear supporting cells and neurons.

Methodology/Principal Findings

We have investigated the role of Prox1 in the developing mouse ear taking advantage of available standard and conditional Prox1 mutant mouse strains using Tg(Pax2-Cre) and Tg(Nes-Cre). A severe reduction in the size of the canal cristae but not of other vestibular organs or the cochlea was identified in the E18.5 Prox1^Flox/Flox; Tg(Pax2-Cre) mutant ear. In these mutant embryos, hair cell differentiated; however, their distribution pattern was slightly disorganized in the cochlea where the growth of type II nerve fibers to outer hair cells along Prox1 expressing supporting cells was severely disrupted. In the case of Nestin-Cre, we found that newborn Prox1^Flox/Flox; Tg(Nestin-Cre) exhibit only a disorganized innervation of outer hair cells despite apparently normal cellular differentiation of the organ of Corti, suggesting a cell-autonomous function of Prox1 in neurons.

Conclusions/Significance

These results identify a dual role of Prox1 during inner ear development; growth of the canal cristae and fiber guidance of Type II fibers along supporting cells in the cochlea.     

Expression of Lhx6 in the adult and developing mouse retina

PurposeTo investigate the expression patterns of LIM Homeobox 6 (Lhx6) in the adult and developing mouse retina.MethodsThe Lhx6-GFP knock-in allele was used to activate constitutive expression of a GFP reporter in Lhx6 expressing cells. Double labeling with GFP and retinal markers in the mouse retina at postnatal day 56 (P56) was performed to identify the cell types expressing Lhx6. To determine the neuronal cell types that express Lhx6, double labeling with GFP and various retinal markers was employed in the differentiating retina at P7 and P15.ResultsGFP + Lhx6 lineage cells were determined in Brn3a + retinal ganglion cells (RGCs), ChAT + amacrine cells (ACs), and Islet-class LIM-homeodomain 1 (Isl1+) ACs in the mouse retina at P56. In the ganglion cell layer (GCL), Lhx6 was expressed in Brn3a + RGCs but not Brn3b + RGCs at P15. Moreover, in the inner nuclear layer (INL), Lhx6 was not expressed in Bhlhb5+ ACs at P15. However, Lhx6 was weakly expressed in Glyt1+ ACs and Pax6+ ACs, and strongly expressed in Isl1+ and ChAT + ACs at P15.ConclusionLhx6 was expressed in RGCs and ACs in both the adult and developing mouse retina.     

Complete reconstruction of the retinal laminar structure from a cultured retinal pigment epithelium is triggered by altered tissue interaction and promoted by overlaid extracellular matrices

The retina regenerates from retinal pigment epithelial (RPE) cells by transdifferentiation in the adult newt and Xenopus laevis when it is surgically removed. This was studied under a novel culture condition, and we succeeded, for the first time, in developing a complete retinal laminar structure from a single epithelial sheet of RPE. We cultured a Xenopus RPE monolayer sheet isolated from the choroid on a filter cup with gels overlaid and found that the retinal tissue structure differentiated with all retinal layers present. In the culture, RPE cells isolated themselves from the culture substratum (filter membrane), migrated, and reattached to the overlaid gel, on which they initiated transdifferentiation. This was exactly the same as observed during in vivo retina regeneration of X. laevis. In contrast, when RPE monolayers were cultured similarly without isolation from the choroid, RPE cells proliferated, but remained pigmented instead of transdifferentiating, indicating that alteration in tissue interaction triggers transdifferentiation. We then examined under the conventional tissue culture condition whether altered RPE‐choroid interaction induces Pax6 expression. Pax6 was upregulated in RPE cells soon after they were removed from the choroid, and this expression was not dependent of FGF2. FGF2 administration was needed for RPE cells to maintain Pax6 expression. From the present results, in addition to our previous ones, we propose a two‐step mechanism of transdifferentiation: the first step is a reversible process and is initiated by the alteration of the cell‐extracellular matrix and/or cell–cell interaction followed by Pax6 upregulation. FGF2 plays a key role in driving RPE cells into the second step, during which they differentiate into retinal stem cells. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009