Expression Patterns Of Pax6 Research Articles

Whole mount immunofluorescence analysis of fresh and stored human donor corneas highlights changes in limbal characteristics during storage

PurposeHuman donor corneas are an essential control tissue for corneal research. We utilized whole mount immunofluorescence (WM-IF) to evaluate how the storage affects the tissue integrity and putative limbal stem cells in human and porcine corneas. Moreover, we compare this information with the marker expression patterns observed in human pluripotent stem cell (hPSC)-derived LSCs. MethodsThe expression of putative LSC markers was analyzed with WM-IF and the fluorescence intensity was quantified in human donor corneas stored for 1–30 days, and in porcine corneas processed 0–6 h after euthanasia. The results were compared with the staining of human and porcine corneal cryosections and with both primary and hPSC-derived LSC cultures. ResultsWM-IF analyses emerged as a more effective method when compared to tissue sections for visualizing the expression of LSC markers within human and porcine corneas. Storage duration was a significant factor influencing the expression of LSC markers, as human tissues stored longer exhibited notable epithelial degeneration and lack of LSC markers. Porcine corneas replicated the expression patterns observed in fresh human tissue. We validated the diverse expression patterns of PAX6 in the limbal-corneal region, which aligned with findings from hPSC-LSC differentiation experiments. ConclusionsWM-IF coupled with quantification of fluorescence intensities proved to be a valuable tool for investigating LSC marker expression in both human and porcine tissues ex vivo. Prolonged storage significantly influences the expression of LSC markers, underscoring the importance of fresh human or substitute control tissue when studying limbal stem cell biology.

转录因子Nkx6.1对脊髓损伤后中间神经元和星形胶质细胞发生的影响

<p indent="0mm">Interneurons are the main local loop connecting sensory and motor neurons and play an important role in the pathological changes after spinal cord injury and the corresponding repair process. After spinal cord injury, astrocyte proliferation and glial scar formation are the main factors hindering the effective regeneration and growth of injured spinal cord axons. In the early stage of neurogenesis, neuroepithelial cells produce astrocytes and intermediate neurons. Therefore, it is likely that astrocytes and intermediate neurons arise from common progenitor cells. In the spinal cord, neuron and glial cell differentiation is regulated by a transcription factor network. In particular, Nkx6.1 is critical for the fate determination of progenitor cells into ventral interneurons. However, the role of Nkx6.1 in the birth of spinal astrocytes after spinal cord injury is still unclear. Herein, we first used the Cre-LoxP system and Rosa26 gene knock-in technology to specifically induce an <italic>Nkx6</italic>.<italic>1</italic> knockout and introduce a lycopene marker gene in different neural precursor cells of adult mice’s spinal cord. Next, we constructed a knockout spinal cord injury model and performed immunofluorescence staining. The staining results showed a substantial number of Nkx6.1⁺ cells in the injured area after spinal cord injury, while <italic>Nkx6</italic>.<italic>1</italic> ablation one week after spinal cord injury would hinder the formation of the glial scar and inhibit the production of En1⁺ intermediate neurons by regulating the expression pattern of Pax6<italic> </italic>in the central neural tube. In conclusion, our study shows that <italic>Nkx6</italic>.<italic>1</italic> plays a crucial role in spinal cord injury, being responsible for effective axon regeneration and motor function recovery.

SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium.

The SARS-CoV-2 pandemic has caused unparalleled disruption of global behavior and significant loss of life. To minimize SARS-CoV-2 spread, understanding the mechanisms of infection from all possible routes of entry is essential. While aerosol transmission is thought to be the primary route of spread, viral particles have been detected in ocular fluid, suggesting that the eye may be a vulnerable point of viral entry. To this end, we confirmed SARS-CoV-2 entry factor and antigen expression in post-mortem COVID-19 patient ocular surface tissue and observed productive viral replication in cadaver samples and eye organoid cultures, most notably in limbal regions. Transcriptional analysis of ex vivo infected ocular surface cells and hESC-derived eye cultures revealed robust induction of NF-κB in infected cells as well as diminished type I/III interferon signaling. Together these data suggest that the eye can be directly infected by SARS-CoV-2 and implicate limbus as a portal for viral entry.

Generation of Pax6-IRES-EGFP knock-in mouse via the cloning-free CRISPR/Cas9 system to reliably visualize neurodevelopmental dynamics

Pax6 encodes a transcription factor that plays pivotal roles in eye development, early brain patterning, neocortical arealization, and so forth. Visualization of Pax6 expression dynamics in these events could offer numerous advantages to neurodevelopmental studies. While CRISPR/Cas9 system has dramatically accelerated one-step generation of knock-out mouse, establishment of gene-cassette knock-in mouse via zygote injection has been considered insufficient due to its low efficiency. Recently, an improved CRISPR/Cas9 system for effective gene-cassette knock-in has been reported, where the native form of guide RNAs (crRNA and tracrRNA) assembled with recombinant Cas9 protein are directly delivered into mouse fertilized eggs. Here we apply this strategy to insert IRES-EGFP-pA cassette into Pax6 locus and achieve efficient targeted insertions of the 1.8 kb reporter gene. In Pax6-IRES-EGFP mouse we have generated, EGFP-positive cells reside in the eyes and cerebellum as endogenous Pax6 expressing cells at postnatal day 2. At the early embryonic stages when the embryos are transparent, EGFP-positive regions can be easily identified without PCR-based genotyping, precisely recapitulating the endogenous Pax6 expression patterns. Remarkably, at E12.5, the graded expression patterns of Pax6 in the developing neocortex now become recognizable in our knock-in mice, serving a sufficiently sensitive and useful tool to precisely visualize neurodevelopmental processes.

Dynamic expression patterns of Pax6 during spermatogenesis in the mouse.

Spermatogenesis is a series of complex processes to generate mature sperm, and various molecules play crucial roles in regulating these processes. Previous studies imply a possibility that a transcriptional factor Pax6, a key player of brain and sensory organ development, could be involved in spermatogenesis, but neither expression nor function of Pax6 in the adult testis has been examined yet. In the present study, we described for the first time Pax6 expression dynamics in the adult mouse testis. Using cell-type-specific markers, the expression of Pax6 was detected in 67.0% of promyelocytic leukemia zinc finger (Plzf)-positive type A spermatogonia. The expression of Pax6 was also observed in p63-positive spermatocytes and round spermatids. We did not detect any expression of Pax6 in Sox9-positive Sertoli cells or in elongated spermatids and mature sperm. High-resolution analyses revealed that Pax6 formed a single dot-like structure during mid-phase of the pachytene spermatocyte. This dot-like structure co-localized with γH2A.X demarcating XY body, a domain in which X and Y chromosomes are silenced and compartmentalized. These results may suggest a novel role of Pax6 in spermatogenesis.

Effects of light and covering behavior on PAX6 expression in the sea urchin Strongylocentrotus intermedius.

We studied the diel expression pattern of PAX6 (a structural gene that is commonly involved in the eye development and photoreception of eye forming animals) and the effects of light and covering behavior on PAX6 expression in the sea urchin Strongylocentrotus intermedius. We confirmed that aphotic condition significantly reduced covering behavior in S. intermedius. The diel expression pattern of PAX6 was significantly different in S. intermedius under photic and aphotic conditions. The gene expression of PAX6 significantly deceased in covered S. intermedius both under natural light and in darkness. The present finding provides valuable insight into the probable link between covering and PAX6 expression of sea urchins. Further studies are required to investigate the detailed expression network of light detection involved genes in order to fully reveal the molecular mechanism of the light-induced covering behavior of sea urchins.

Expression patterns of Pax6 and Pax7 in the adult brain of a urodele amphibian, Pleurodeles waltl

Expression patterns of Pax6, Pax7, and, to a lesser extent, Pax3 genes were analyzed by a combination of immunohistochemical techniques in the central nervous system of adult specimens of the urodele amphibian Pleurodeles waltl. Only Pax6 was found in the telencephalon, specifically the olfactory bulbs, striatum, septum, and lateral and central parts of the amygdala. In the diencephalon, Pax6 and Pax7 were distinct in the alar and basal parts, respectively, of prosomere 3. The distribution of Pax6, Pax7, and Pax3 cells correlated with the three pretectal domains. Pax7 specifically labeled cells in the dorsal mesencephalon, mainly in the optic tectum, and Pax6 cells were the only cells found in the tegmentum. Large populations of Pax7 cells occupied the rostral rhombencephalon, along with lower numbers of Pax6 and Pax3 cells. Pax6 was found in most granule cells of the cerebellum. Pax6 cells also formed a column of scattered neurons in the reticular formation and were found in the octavolateral area. The rhombencephalic ventricular zone of the alar plate expressed Pax7. Dorsal Pax7 cells and ventral Pax6 cells were found along the spinal cord. Our results show that the expression of Pax6 and Pax7 is widely maintained in the brains of adult urodeles, in contrast to the situation in other tetrapods. This discrepancy could be due to the generally pedomorphic features of urodele brains. Although the precise role of these transcription factors in adult brains remains to be determined, our findings support the idea that they may also function in adult urodeles.

Stimulation of mouse Cyp1b1 during adipogenesis: Characterization of promoter activation by the transcription factor Pax6

Cytochrome P4501B1 (Cyp1b1) is expressed specifically in certain neural crest (NC) cells during embryogenesis. Mesenchymal progenitor cells that develop from NC cells are modeled here by mouse C3H10T1/2 and 3T3-L1 cells. Dexamethasone in combination with methylisobutylxanthine (DM) induces Cyp1b1 and a 6.7kb mouse Cyp1b1 promoter-luciferase reporter in each cell type prior to adipogenesis. An 18 base sequence (at −6.11kb) (PaxE) which was essential for this reporter stimulation in 3T3-L1 cells bound the transcription factor Pax6. This is shown by gel mobility shifts and sequence mutations. Heterologous vector expression of Pax6 in 3T3-L1 cells enhanced DM stimulated Cyp1b1 promoter activity through cooperation with two Sp1 sites in the proximal promoter region. Chromatin immunoprecipitation showed that DM stimulated binding of Pax6 adjacent to Sp1 in the proximal promoter more than in the PaxE region. The Cyp1b1 induction by DM in C3H10T1/2 cells was more rapid but independent of Pax6. The far upstream enhancer region (FUER) found in rat Cyp1b1 responded to DM but was inactive in the mouse promoter due to key sequence changes. The expression patterns of Pax6 and Cyp1b1 frequently overlap during mouse embryogenesis. The relationship between Pax6 and Cyp1b1 expression warrants further investigation, particularly in the NC.

Dynamic expression of Pax6 in the shark olfactory system: evidence for the presence of Pax6 cells along the olfactory nerve pathway

Pax6 is involved in the control of neuronal specification, migration, and differentiation in the olfactory epithelium and in the generation of different interneuron subtypes in the olfactory bulb. Whether these roles are conserved during evolution is not known. Cartilaginous fish are extremely useful models for assessing the ancestral condition of brain organization because of their phylogenetic position. To shed light on the evolution of development of the olfactory system in vertebrates and on the involvement of Pax6 in this process, we analyzed by in situ hybridization and immunohistochemistry the expression pattern of Pax6 in the developing olfactory system in a basal vertebrate, the lesser spotted dogfish Scyliorhinus canicula. This small shark is becoming an important fish model in studies of vertebrate development. We report Pax6 expression in cells of the olfactory epithelium and olfactory bulb, and present the first evidence in vertebrates of strings of Pax6-expressing cells extending along the developing olfactory nerve. The results indicate the olfactory epithelium as the origin of these cells. These data are compatible with a role for Pax6 in the development of the olfactory epithelium and fibers, and provide a basis for future investigations into the mechanisms that regulate development of the olfactory system throughout evolution.

Spatiotemporal expression patterns of Pax6 in the brain of embryonic, newborn, and adult mice

The transcription factor Pax6 has been reported to specify neural progenitor cell fates during development and maintain neuronal commitments in the adult. The spatiotemporal patterns of Pax6 expression were examined in sagittal and horizontal sections of the embryonic, postnatal, and adult brains using immunohistochemistry and double immunolabeling. The proportion of Pax6-immunopositive cells in various parts of the adult brain was estimated using the isotropic fractionator methodology. It was shown that at embryonic day 11 (E11) Pax6 was robustly expressed in the proliferative neuroepithelia of the ventricular zone in the forebrain and hindbrain, and in the floor and the mesencephalic reticular formation (mRt) in the midbrain. At E12, its expression emerged in the nucleus of the lateral lemniscus in the rhombencephalon and disappeared from the floor of the midbrain. As neurodevelopment proceeds, the expression pattern of Pax6 changes from the mitotic germinal zone in the ventricular zone to become extensively distributed in cell groups in the forebrain and hindbrain, and the expression persisted in the mRt. The majority of Pax6-positive cell groups were maintained until adult life, but the intensity of Pax6 expression became much weaker. Pax6 expression was maintained in the mitotic subventricular zone in the adult brain, but not in the germinal region dentate gyrus in the adult hippocampus. There was no obvious colocalization of Pax6 and NeuN during embryonic development, suggesting Pax6 is found primarily in developing progenitor cells. In the adult brain, however, Pax6 maintains neuronal features of some subtypes of neurons, as indicated by 97.1% of Pax6-positive cells co-expressing NeuN in the cerebellum, 40.7% in the olfactory bulb, 38.3% in the cerebrum, and 73.9% in the remaining brain except the hippocampus. Differentiated tyrosine hydroxylase (TH) neurons were observed in the floor of the E11 midbrain where Pax6 was also expressed, but no obvious colocaliztion of TH and Pax6 was detected. No Pax6 expression was observed in TH-expressing areas in the midbrain at E12, E14, and postnatal day 1. These results support the notion that Pax6 plays pivotal roles in specifying neural progenitor cell commitments and maintaining certain mature neuronal fates.

Significance of the Dynamic Expression of Pax6 and Dlx2 in the Postnatal Periventricular Zone Cells

Significance of the Dynamic Expression of Pax6 and Dlx2 in the Postnatal Periventricular Zone Cells

Aging-associated modulation in the expression of Pax6 in mouse brain.

Symptoms like mental retardation, depression, and anxiety have been observed during aging. Almost similar phenotypes have been evident in patients having haploinsufficiency or mutations in Pax6, a transcriptional regulator. Since Pax6 regulates axon guidance, differentiation of neurons from glia, and neuronal migration, it has been considered as a marker of newly generated neurons. The immunohistochemical analysis of Pax6 positive cells and expression pattern of Pax6 in olfactory lobe, hippocampus, and cerebellum of aging mouse brain have been investigated. The number of Pax6 positive cells and level of Pax6 were reduced progressively in olfactory lobe, cerebellum, and hippocampus from postnatal day-zero (P0) to old age mice. Pax6 positive cells were significantly lower in dentate gyrus, CA1, CA2, and CA3 regions of hippocampus, in mitral cell (MiCe), and internal plexiform (InPl) layers of olfactory lobe, and in granular cell (GrLa), and Purkinje's cell (PuCe) layers of cerebellum from P0 to old age. Thus, modulation in the expression of Pax6 and reduction in Pax6 positive cells show direct association of Pax6 with aging-related neuronal dystrophy.

In Vitro Modelling of Cortical Neurogenesis by Sequential Induction of Human Umbilical Cord Blood Stem Cells

Neurogenesis of excitatory neurons in the developing human cerebral neocortex is a complex and dynamic set of processes and the exact mechanisms controlling the specification of human neocortical neuron subtypes are poorly understood due to lack of relevant cell models available. It has been shown that the transcription factors Pax6, Tbr2 and Tbr1, which are sequentially expressed in the rodent neocortex, regulate and define corticogenesis of glutamatergic neocortical neurons. In humans the homologues of these genes are generally expressed in a similar pattern, but with some differences. In this study, we used purified human umbilical cord blood stem cells, expressing pluripotency marker genes (OCT4, SOX2 and NANOG), to model human neocortical neurogenesis in vitro. We analyzed the expression patterns of PAX6, TBR2 and TBR1, at both protein and mRNA levels, throughout the 24 days of a sequential neuronal induction protocol. Their expression patterns correlated with those found in the developing human neocortex where they define different developmental stages of neocortical neurons. The derived cord blood neuron-like cells expressed a number of neuronal markers. They also expressed components of glutamatergic neurotransmission including glutamate receptor subunits and transporters, and generated calcium influxes upon stimulation with glutamate. Thus we have demonstrated that it is possible to model neocortical neurogenesis using cord blood stem cells in vitro. This may allow detailed analysis of the molecular mechanisms regulating neocortical neuronal specification, thus aiding the development of potential therapeutic tools for diseases and injuries of the cerebral cortex.

Regionalization of the Shark Hindbrain: A Survey of an Ancestral Organization

Cartilaginous fishes (chondrichthyans) represent an ancient radiation of vertebrates currently considered the sister group of the group of gnathostomes with a bony skeleton that gave rise to land vertebrates. This out-group position makes chondrichthyans essential in assessing the ancestral organization of the brain of jawed vertebrates. To gain knowledge about hindbrain evolution we have studied its development in a shark, the lesser spotted dogfish Scyliorhinus canicula by analyzing the expression of some developmental genes and the origin and distribution of specific neuronal populations, which may help to identify hindbrain subdivisions and boundaries and the topology of specific cell groups. We have characterized three developmental periods that will serve as a framework to compare the development of different neuronal systems and may represent a suitable tool for comparing the absolute chronology of development among vertebrates. The expression patterns of Pax6, Wnt8, and HoxA2 genes in early embryos of S. canicula showed close correspondence to what has been described in other vertebrates and helped to identify the anterior rhombomeres. Also in these early embryos, the combination of Pax6 with protein markers of migrating neuroblasts (DCX) and early differentiating neurons (general: HuC/D; neuron type specific: GAD, the GABA synthesizing enzyme) revealed the organization of S. canicula hindbrain in both transverse segmental units corresponding to visible rhombomeres and longitudinal columns. Later in development, when the interrhombomeric boundaries fade away, accurate information about S. canicula hindbrain subdivisions was achieved by comparing the expression patterns of Pax6 and GAD, serotonin (serotoninergic neurons), tyrosine hydroxylase (catecholaminergic neurons), choline acetyltransferase (cholinergic neurons), and calretinin (a calcium-binding protein). The patterns observed revealed many topological correspondences with other vertebrates and led to reconsideration of the current view of the elasmobranch hindbrain segmentation as peculiar among vertebrates.

Dynamic expression of Pax6 in the shark olfactory system: evidence for the presence of Pax6 cells along the olfactory nerve pathway

Pax6 is involved in the control of neuronal specification, migration, and differentiation in the olfactory epithelium and in the generation of different interneuron subtypes in the olfactory bulb. Whether these roles are conserved during evolution is not known. Cartilaginous fish are extremely useful models for assessing the ancestral condition of brain organization because of their phylogenetic position. To shed light on the evolution of development of the olfactory system in vertebrates and on the involvement of Pax6 in this process, we analyzed by in situ hybridization and immunohistochemistry the expression pattern of Pax6 in the developing olfactory system in a basal vertebrate, the lesser spotted dogfish Scyliorhinus canicula. This small shark is becoming an important fish model in studies of vertebrate development. We report Pax6 expression in cells of the olfactory epithelium and olfactory bulb, and present the first evidence in vertebrates of strings of Pax6-expressing cells extending along the developing olfactory nerve. The results indicate the olfactory epithelium as the origin of these cells. These data are compatible with a role for Pax6 in the development of the olfactory epithelium and fibers, and provide a basis for future investigations into the mechanisms that regulate development of the olfactory system throughout evolution. J. Exp. Zool. (Mol. Dev. Evol.) 318:79–90, 2012. © 2011 Wiley Periodicals, Inc.

Mutation in a Novel Connexin-like Gene (Gjf1) in the Mouse Affects Early Lens Development and Causes a Variable Small-Eye Phenotype

The purpose of the study was the characterization of the novel small-eye mutant Aey12 in the mouse. The eyes of the mutants were described morphologically and histologically and by in situ hybridization. The homozygotes were viable and fully fertile, which identifies Aey12 as a new microphthalmia phenotype in the mouse, different from Maf or Pax6 mutants. Histologic analysis indicated the presence of the lens vesicle; however, the primary fiber cells did not elongate properly. Genome-wide linkage analysis mapped the mutation to the proximal region of chromosome 10 between the markers D10Mit206 and D10Mit189. Among the positional candidate genes, one EST (expressed sequence tag), D230044M03Rik, encodes a connexin-like protein. A G-->T point mutation was identified at cDNA position 96, resulting in an R32Q amino acid exchange in a transmembrane domain. The mutation leads to a loss of an SsiI restriction site, which is present in five wild-type mouse strains (102, C3H, C57BL/6, DBA, and JF1). The gene is expressed in the posterior part of the lens vesicle, where the primary fiber elongation starts. In the mutants, the expression pattern of Pax6, Prox1, Six3, and Crygd are modified, but not the pattern of Pax2. The mutated mouse gene belongs to the family of connexin-encoding genes (gene symbols Gja-Gje). Together with its rat and human homologues, it defines a new subgroup, referred to as Gjf1. The mouse mutant described herein offers a new functional candidate gene for microphthalmia-related disorders at the corresponding locus on human chromosome 6, area q24.

Transcription factor protein expression patterns by neural or neuronal progenitor cells of adult monkey subventricular zone

The anterior subventricular zone of the adult mammalian brain contains progenitor cells which are upregulated after cerebral ischemia. We have previously reported that while a part of the progenitors residing in adult monkey anterior subventricular zone travels to the olfactory bulb, many of these cells sustain location in the anterior subventricular zone for months after injury, exhibiting a phenotype of either neural or neuronal precursors. Here we show that ischemia increased the numbers of anterior subventricular zone progenitor cells expressing developmentally regulated transcription factors including Pax6 (paired-box 6), Emx2 (empty spiracles-homeobox 2), Sox 1–3 (sex determining region Y-box 1–3), Ngn1 (neurogenin 1), Dlx1,5 (distalless-homeobox 1,5), Olig1,3 (oligodendrocyte lineage gene 1,3) and Nkx2.2 (Nk-box 2.2), as compared with control brains. Analysis of transcription factor protein expression by sustained neural or neuronal precursors in anterior subventricular zone revealed that these two cell types were positive for characteristic sets of transcription factors. The proteins Pax6, Emx2, Sox2,3 and Olig1 were predominantly localized to dividing neural precursors while the factors Sox1, Ngn1, Dlx1,5, Olig2 and Nkx2.2 were mainly expressed by neuronal precursors. Further, differences between monkeys and non-primate mammals emerged, related to expression patterns of Pax6, Olig2 and Dlx2. Our results suggest that a complex network of developmental signals might be involved in the specification of primate progenitor cells.

Pax-6 expression and activity are induced in the reepithelializing cornea and control activity of the transcriptional promoter for matrix metalloproteinase gelatinase B

Recent evidence supports the idea that matrix metalloproteinases (MMPs) act as morphogenetic regulators in embryonic and adult events of tissue remodeling. MMP activity is controlled primarily at the level of gene expression. In a recent study we characterized the transcriptional promoter of the MMP gene, gelatinase B (gelB), in transgenic mice, demonstrating the requirement for DNA sequences between -522 and +19 for appropriate activity. In this study we investigated factors required for gelB promoter activity in the developing eye and reepithelializing adult cornea. Pax-6 is a homeobox and paired domain transcription factor that acts at the top of the hierarchy of genes controlling eye development. Pax-6 is also expressed in the adult eye. We show here that the tissue expression pattern of Pax-6 overlaps extensively with gelB promoter activity in the developing and adult eye. In addition Pax-6 is observed to be upregulated in repairing corneal epithelium, as is gelB promoter activity. In cell culture transfection experiments, we identified two promoter regions which mediate positive response to Pax-6. By electrophoretic mobility shift assay, we further pinpoint two Pax-6 binding sites within these response regions and demonstrate direct interaction of the Pax-6 paired domain with one of these sites. These data suggest a mechanism by which Pax-6 may direct gelB expression in an eye-specific manner.

Pax6 defines the di-mesencephalic boundary by repressing En1 and Pax2.

Transcriptional factors and signaling molecules are responsible for regionalization of the central nervous system. In the early stage of neural development, Pax6 is expressed in the prosencephalon, while En1 and Pax2 are expressed in the mesencephalon. Here, we misexpressed Pax6 in the mesencephalon to elucidate the mechanism of the di-mesencephalic boundary formation. Histological analysis, expression patterns of diencephalic marker genes, and fiber trajectory of the posterior commissure indicated that Pax6 misexpression caused a caudal shift of the di-mesencephalic boundary. Pax6 repressed En1, Pax2 and other tectum (mesencephalon)-related genes such as En2, Pax5, Pax7, but induced Tcf4, a diencephalon marker gene. To know how Pax6 represses En1 and Pax2, we ectopically expressed a dominant-active or negative form of Pax6. The dominant-active form of Pax6 showed a similar but more severe phenotype than Pax6, while the dominant-negative form showed an opposite phenotype, suggesting that Pax6 acts as a transcriptional activator. Thus Pax6 may repress tectum-related genes by activating an intervening repressor. The results of misexpression experiments, together with normal expression patterns of Pax6, En1 and Pax2, suggest that repressive interaction between Pax6 and En1/Pax2 defines the di-mesencephalic boundary.

Pax-6 expression during retinal regeneration in the adult newt.

The present study examined the expression of Pax-6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax-6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax-6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax-6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax-6 became similar to that of a normal retina. It was found that Pax-6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax-6 changed as cell differentiation proceeded during retinal regeneration.