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Mutations in mammalian Pax-6 homologs: Pax-6 role in eye development

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance. The expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, it has been demonstrated that the addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, the expression of several early lens placode-specific markers were examined in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway that is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. It is concluded that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation (Wawersik 1999).

The Pax6 transcription factor plays a key role in ocular development of vertebrates and invertebrates. Homozygosity of the Pax6 null mutation in human and mice results in arrest of optic vesicle development and failure to initiate lens formation. This phenotype obscures the understanding of autonomous function of Pax6 in these tissue components and during later developmental stages. The Cre/loxP approach was employed to inactivate Pax6 specifically in the eye surface ectoderm concomitantly with lens induction. Although lens induction occurs in the mutant, as indicated by Sox2 (Drosophila homolog SoxNeuro: Cremazy, 2000) up-regulation in the surface ectoderm, further development of the lens is arrested. Hence, Pax6 activity was found to be essential in the specified ectoderm for lens placode formation. Furthermore, this mutant model allowed an in vivo assessment of the development of a completely normal retina in the absence of early lens structures. Remarkably, several independent, fully differentiated neuroretinas developed in a single optic vesicle in the absence of a lens, demonstrating that the developing lens is not necessary to instruct the differentiation of the neuroretina but is, rather, required for the correct placement of a single retina in the eye (Ashery-Padan, 2000).

The current model for lens induction in vertebrates suggests that the prospective lens ectoderm is determined in a series of sequential steps. Initially, during the late gastrula stage of the amphibian embryo, the competence of the ectoderm to respond to the induction signals is acquired. Subsequently, planar signals from the anterior neural plate induce the lens-forming bias in the head ectoderm. Finally, the contact with the underlying optic vesicle (OV) triggers the specification of the lens ectoderm leading to changes in gene expression during the preplacode stage. These changes are followed by the formation of the lens placode (Ashery-Padan, 2000 and references therein).

In vertebrates, Otx2, BMP4, and Pax6 have been implicated to play parallel roles in maintaining the competence of the surface ectoderm (SE) to respond to the signals from the OV. Several lines of evidence pointed to the importance of Pax6 in this process: (1) early expression of Pax6 in the ectoderm coincides with the lens-competence stage; (2) tissue recombination experiment has demonstrated that Pax6-/- ectoderm rather than Pax6-/- OV is responsible for the lens defects in Pax6-/- mice; (3) Pax6 chimera analysis has shown that Pax6 is required before formation of the lens placode. Furthermore, markers indicative of lens specification such as Sox2 and Frissled-related protein (sFRP2) are not expressed in the SE of Pax6-/- embryos. This apparent requirement for Pax6 for the initial response of the ectoderm hampered earlier studies aimed at understanding the role and identifying the downstream targets of Pax6 after lens specification. However, in the Le-mutant, Pax6 expression occurs early and is switched off, specifically in the SE, before placode formation. The analyses of Le-mutant reveal that Pax6 is required for the transition from lens specification to lens placode (Ashery-Padan, 2000 and references therein).

Up-regulation of Sox2 is one of the first responses of the specified ectoderm to the inductive signals; Sox2 expression precedes lens placode formation, and its expression is dependent on signals from the OV. Furthermore, Sox2 is not up-regulated in the SE of Pax6, BMP4, or BMP7 mutant embryos, in which lens induction is impaired. In the Le-mutant embryos, the early, normal expression of Pax6 in the eye is sufficient for the up-regulation of Sox2. These results demonstrate that up-regulation of Sox2 is dependent on Pax6, while continuous Pax6 activity in the SE is not required for the maintenance of Sox2 expression. By employing Le-mutant, whether Pax6 in the responding SE or Pax6 in the inducing OV mediates the initial up-regulation of Sox2 cannot be defined. The Le-mutant nevertheless allows studies to address the autonomous function of Pax6 in the SE following lens specification during the formation of the lens placode (Ashery-Padan, 2000 and references therein).

The preplacode stage precedes the appearance of the lens placode. During this stage, feedback signals are required to maintain the expression of Pax6 and other genes in the ectoderm. BMP7 has been implicated in playing a role in this step. The expression of Six3 (homolog of the Drosophila sine-oculis gene) and Prox1 (homolog of the Drosophila prospero gene) have been reported to coincide with the lens placode stage. In this article, it is demonstrated that Pax6 is essential in the preplacode for lens placode formation. This conclusion is based on the following three observations: (1) in the Le-mutant, the lens placode does not form; (2) the transcriptional activity of the Le-Cre transgene is not maintained; (3) the expression of Six3 and Prox1 in the specified ectoderm is absent and, thus, dependent on Pax6 (Ashery-Padan, 2000 and references therein).

The dependence on Pax6 function of several regulatory genes after lens formation probably accounts for the complete arrest in lens development observed in Le-mutant. The role of Six3 is currently unknown; however, members of the vertebrate Six genes, HSIX1 and XOptx2, have been shown to affect cell proliferation. It is possible that loss of Six3 in the SE causes a failure in the onset of proliferation, which is required for lens placode formation. Prox1 has been recently shown to play an important role in lens fiber elongation, while Pax6 has been inferred to play a direct role in regulation of crystalline expression. Thus, probably both Prox1 and Pax6 are essential during later stages of lens development (Ashery-Padan, 2000 and references therein).

Vertebrate gene families, homologous to the Drosophila eye-determination genes, have been identified recently, and the regulatory interactions between them seem to be conserved and redeployed during vertebrate somite development. The complete dependence of Six3 expression on Pax6 activity in the specified ectoderm of the Le-mutant is similar to the reported dependence of so on eye expression described in Drosophila. This result implies that the regulatory hierarchy between Pax6 and Six3 in the SE during the preplacode stage is conserved in evolution. In the OV, however, these genes seem to function in parallel pathways because the expression of the vertebrate so homologs Six3 and Six6/optx2 is maintained in the Pax6-/- mice (Ashery-Padan, 2000 and references therein).

Existence of a positive feedback loop for maintaining Pax6 expression during the preplacode stage has been proposed based on the BMP7-/- phenotype. Furthermore, loss of ectodermal Pax6 expression during lens placode formation was observed. In line with these results, it was observed that the Le-Cre transgene is not transcriptionally active in the Le-mutant SE after E9.5. This indicates that the transcription activity of the Pax6 regulatory regions in the transgene depends on Pax6, and thus, confirms the requirement for an autoregulatory feedback loop. In Drosophila, so participates in a positive feedback loop to maintain eye expression in the eye disc. Therefore, it is possible that Six3 in the preplacode is part of a regulatory loop mediating the maintenance of Pax6 expression. Indeed, up-regulation of Pax6 on misexpression of Six3 has been demonstrated in transgenic mice, and Six3 binding sites have been identified on the Pax6 regulatory sequences employed in the Le-Cre transgene (Ashery-Padan, 2000 and references therein).

The influence of the lens, after the lens vesicle stage, on the development of the retina has been studied by mechanical ablation of the lens. In these studies, convolutions of the retina were observed. These folds were attributed to change in ocular pressure, and it was concluded that following the formation of the optic cup and the lens vesicle, retinal development is mostly independent from the lens. The roles of the early signals emanating from the lens primordium to the OV were addressed by early experiments of classical embryologists and in recent work in chicks. Two main functions were attributed to the early signals: delineation of the prospective NR and the RPE domains from the bipotential population of the OV precursors, and mediation of the parallel invaginations of the two tissues to form the lens vesicle and the optic cup (Ashery-Padan, 2000 and references therein).

There are only a few mouse mutants, including Pax6, BMP7, and BMP4, in which the absence of the lens placode has been reported. In these mutants, as well as in the Pax6 chimera studies, the influence of the SE on the development of the OV could not be directly addressed, mainly because of the direct requirement for these genes in both the inducting OV, as well as in the responding SE. The absence of a lens placode in the conditional Le-mutant mice exposed the in vivo role of the earliest lens structures in retina patterning, morphology, and differentiation (Ashery-Padan, 2000 and references therein).

The delay in invagination of the OV in the E10 Le-mutant eye is the first obvious morphological phenotype of the retina. This phenotype reveals the essential role of the lens placode for optic cup formation. Despite this morphological change, the initial distribution of Trp2 and Chx10 transcripts in the OV suggests that the separation of the bipotential population of cells in the OV to the prospective RPE and NR domains occurs in the Le-mutant. This result supports the view that the progenitor domains in the OV are established independently from the lens placode and possibly before the placode is formed (Ashery-Padan, 2000 and references therein).

At subsequent stages of development (E11), several invaginations were observed in the Le-mutant OV. These retina folds seem to form separate retina subcompartments based on the histology of the Le-mutant eyes, the distribution of Pax6 and TUJI staining in each retina fold, and the expression of Trp2 between the folds. Moreover, some of the NR domains evolved in proximal regions that opposed the mesenchyme, while RPE differentiated from distal parts of the OV. This suggests that a change in progenitor cell fate has occurred in these regions (Ashery-Padan, 2000 and references therein).

There are several possible explanations for the formation of the retina subcompartments in Le-mutant eyes: the signals from the lens might influence the pattern of cell proliferation or the timing of retina cell differentiation. Both alternations would result in a change in the distribution and/or an increase of overall cell number in the retina, leading to irregular folding of the retina within the limited space of the eye. Changes in ocular pressure might also contribute to the extensive folding of the retina, as suggested by the early works on lens ablation. Such folds might result in subcompartments that give rise to multiple retinas. Change in retinal cell fate has been reported to occur in vertebrates before cup formation, either because of changes in ocular pressure or as a result of disturbance of contact between the NR and RPE. The delay in optic cup formation in Le-mutant eye might, therefore, result in subsequent changes in progenitor cell fate leading to apparent patches of NR and RPE domains (Ashery-Padan, 2000 and references therein).

Following the invagination, each retina fold seems to develop autonomously. In each fold, a central to peripheral pattern of neuronal cell differentiation was observed, similar to the pattern of neuronal cell differentiation in normal retina. In postnatal Le-mutant eyes, the different retinal neuronal cell types are detected and the neuroretina laminates. Hence, the capacity to form a multilayered retina is autonomous to each fold and is independent of any lens structures (Ashery-Padan, 2000 and references therein).

A scheme summarizing the possible roles of Pax6 in the SE leading to lens placode formation is presented. Three steps precede the formation of the lens placode: lens competence, lens specification, and the preplacode (maintenance) stage. The competence of the ectoderm to respond to the inductive signals from the OV is mediated by Pax6. During lens induction, the upregulation of Sox2 is dependent on Pax6 activity. Inactivation of Pax6 in Le-mutant occurs concomitantly with lens induction and after the up-regulation of Sox2. The expression of lens specific genes, Six3 and Prox1, in the preplacode stage is dependent on Pax6 activity. Feedback signals possibly mediated by six3 are required to maintain Pax6 expression in the SE. The regulatory genes promote lens placode formation and subsequent lens differentiation (Ashery-Padan, 2000).

Aniridia in man and Small eye in mice are semidominant developmental disorders caused by mutations within the paired box gene PAX6. Whereas heterozygotes suffer from iris hypoplasia, homozygous mice lack eyes and nasal cavities and exhibit brain abnormalities. Yeast artificial chromosome transgenic mice have been created carrying the human PAX6 locus. When crossed onto the Small eye background, the transgene rescues the mutant phenotype. Strikingly, mice carrying multiple copies on a wild-type background show specific developmental abnormalities of the eye, but not of other tissues expressing the gene. Thus, at least five different eye phenotypes are associated with changes in PAX6 expression. Thus, not only reduced, but also increased levels of transcriptional regulators can cause developmental defects (Schedl, 1996).

Math5, a mouse basic helix-loop-helix (bHLH) gene that is closely related to Drosophila atonal and Xenopus Xath5, is largely restricted to the developing eye. Math5 retinal expression precedes differentiation of the first neurons and persists within progenitor cells until after birth. To position Math5 in a hierarchy of retinal development, Math5 and Hes1 expression were compared in wild-type and Pax6-deficient (Sey) embryos. Math5 expression is downregulated in Sey/+ eyes and abolished in Sey/Sey eye rudiments, whereas the bHLH gene Hes1 is upregulated in a similar dose-dependent manner. These results link Pax6 to the process of retinal neurogenesis and provide the first molecular correlate for the dosage-sensitivity of the Pax6 phenotype. During retinogenesis, Math5 is expressed significantly before NeuroD, Ngn2 or Mash1. To test whether these bHLH genes influence the fates of distinct classes of retinal neurons, Math5 and Mash1 were ectopically expressed in Xenopus retinal progenitors. Unexpectedly, lipofection of either mouse gene into the frog retina causes an increase in differentiated bipolar cells. Directed expression of Math5, but not Xath5, in Xenopus blastomeres produces an expanded retinal phenotype. It is proposed that Math5 acts as a proneural gene, but has properties different from its most closely related vertebrate family member, Xath5 (Brown, 1998).

Chimeric mice were made by aggregating Pax6 -/- and wild-type mouse embryos, in order to study the interaction between the optic vesicle and the prospective lens epithelium during early stages of eye development. Histological analysis of the distribution of homozygous mutant cells in the chimeras shows that the cell-autonomous removal of Pax6 -/- cells from the lens is nearly complete by E9.5. Most mutant cells are eliminated from an area of facial epithelium wider than, but including, the developing lens placode. This result suggests a role for Pax6 in maintaining a region of the facial epithelium that has the tissue competence to undergo lens differentiation. Segregation of wild-type and Pax6 -/- cells occurs in the optic vesicle at E9.5 and is most likely a result of different adhesive properties of wild-type and mutant cells. Also, proximo-distal specification of the optic vesicle (as assayed by the elimination of Pax6 -/- cells distally), is disrupted in the presence of a high proportion of mutant cells. This suggests that Pax6 operates during the establishment of patterning along the proximo-distal axis of the vesicle. Examination of chimeras with a high proportion of mutant cells shows that Pax6 is required in the optic vesicle for maintenance of contact with the overlying lens epithelium. This may explain why Pax6 -/- optic vesicles are inefficient at inducing a lens placode. Contact is preferentially maintained when the lens epithelium is also wild-type. Together, these results demonstrate requirements for functional Pax6 in both the optic vesicle and surface epithelia in order to mediate the interactions between the two tissues during the earliest stages of eye development (Collinson, 2000).

There is a cell-autonomous requirement for Pax6 in the formation of the lens in low-percentage chimeras. This cell-autonomous requirement is manifest at E9.5 during the earliest stages of placode formation. The unexpected loss of Pax6 minus cells from an area that is wider than the lens placode, before the placode develops, suggests that there is a very early requirement in the broad area of Pax6-expressing pre-placodal head ectoderm. This is consistent with a role for Pax6 in maintaining those cells that have the properties of classical lens competence or bias, representing stages in the lens formation pathway that take place prior to appearance of the lens placode (Collinson, 2000 and references therein).

The roles of Pax6 were investigated in the murine eye and the olfactory epithelium by analyzing 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 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 can 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 behavior of mutant cells in other developmental contexts, particularly the lens, suggesting that common primary defects may be responsible for diverse Pax6-related phenotypes (Collinson, 2003).

The molecular basis of the Pax2 and Pax6 function in the establishment of visual system territories has been studied. Loss-of-function mutants have revealed crucial roles for Pax2 in the generation of the optic stalk and for Pax6 in the development of the optic cup. Ectopic expression of Pax6 in the optic stalk under control of Pax2 promoter elements results in a shift of the optic cup/optic stalk boundary, indicated by the presence of retinal pigmented cells on the optic stalk. By studying mouse embryos at early developmental stages an expansion of Pax2 expression domain was detected in the Pax6-/- mutant and an expansion of the Pax6 expression domain in the Pax2-/- embryo. These results suggest that the position of the optic cup/optic stalk boundary depends on Pax2 and Pax6 expression, hinting at a possible molecular interaction. Using gel shift experiments, the presence was confirmed of Pax2- and Pax6- binding sites on the retina enhancer of the Pax6 gene and on the Pax2 upstream control region, respectively. Co-transfection experiments have revealed a reciprocal inhibition of Pax2 promoter/enhancer activity by Pax6 protein and vice versa. Based on these findings, a model for Pax gene regulation that establishes the proper spatial regionalization of the mammalian visual system is proposed (Schwartz, 2000).

The following molecular model for visual system regionalization is proposed. The signaling molecule Sonic hedgehog, which establishes the midline of the brain and subdivides the eye domain, is presumably the initial activator of Pax2. Strong evidence comes from analysis in mice lacking sonic hedgehog function. With regard to eye patterning, it has been demonstrated that sonic hedgehog is required for Pax2 expression and optic stalk formation. These results are consistent with previous evidence from zebrafish studies suggesting that sonic hedgehog activity from the ventral midline normally stimulates expression of Pax2 in the adjacent optic stalk precursors and represses expression of Pax6. This in turn restricts Pax6 expression to distal portions of the optic vesicle. Unfortunately, no candidate gene activating Pax6 in the prosencephalic portion of the neural plate has been described. A possible candidate directly or indirectly acting on Pax6 activation, based on the spatial-temporal expression pattern, could be the homeobox protein Otx2 or the cell-signaling molecule Notch1 (Schwartz, 2000 and references therein).

After their initial activation, the Pax2 and Pax6 expression becomes independent of the activating factors, owing to their autocatalytic enhancement. Pax6 protein can bind its own enhancer and has the potential to stimulate transcription. The Pax2 protein, in turn, has the capacity to bind to the Pax6 enhancer and represses transcription. The Pax6 protein has the same function on the Pax2 enhancer. Although the Pax2 promoter region contains one site for Pax2 protein binding, no Pax2 autocatalytic enhancement was observed in the cell culture assays. Sonic hedgehog could be hypothesized to be the Pax2 activator, since the sonic hedgehog gradient always reaches the Pax2-expressing territory; alternatively, the promoter region used in the transfection experiment does not contain the element(s) that are essential for Pax2 self-activation. Consequently, in a region where the activity of these genes overlaps, reciprocal inhibition establishes a boundary, finally leading to a steady state where no cells express both proteins at the same time. This in consequence leads to the formation of the optic stalk and optic cup boundary. However, it is not known whether the Pax2 and Pax6 boundary and therefore the division of a Pax2- and a Pax6- positive domain (which follow very different developmental fates) is the first separation generating positional information for the eye field (Schwartz, 2000 and references therein).

In Drosophila, similar to the vertebrate system where Pax2 expression is restricted to the optic stalk and Pax6 expression to the optic cup and the lens, sparkling and eyeless are expressed in the homolog structures. sparkling expression is found in the precursors of cone and primary pigment cells, whereas eyeless expression is restricted to regions anterior to the morphogenetic furrow in the undifferentiated part of the eye disc epithelium. In addition, the phenotype of the eyeless or the sparkling mutation is comparable to the Pax6-/- and Pax2-/- phenotypes in the mouse. In conclusion, Pax2 and Pax6 and their Drosophila homologs sparkling and eyeless, play important and strikingly conserved roles in the morphogenesis and regional specification of the vertebrate or insect eyes.The visual system is most probably not the only example of reciprocal inhibition between Pax genes in the regionalization of the body plan. These genes are expressed in adjacent territories in other embryonic structures. The regionalization of the midbrain/forebrain boundary is crucially dependant upon Pax6 expression in the prosencephalon, and on Pax2 and Pax5 expression in the mesencephalon. In the spinal cord, Pax1/9 and Pax3/7 could be good candidates for such a regionalization mechanism in more caudal parts of the neuroectoderm. Therefore, the molecular mechanism described here for the eye may be of more general importance (Schwartz, 2000).

Pax6 is a member of the mammalian Pax transcription factor family. Many of the Pax genes display semi-dominant loss-of-function heterozygous phenotypes, yet the underlying cause for this dosage requirement is not known. Mice heterozygous for Pax6 mutations exhibit small eyes (Sey) and in embryos the most obvious defect is a small lens. Lens development has been studied in Pax6Sey-1Neu/+ embryos to understand the basis of the haploinsufficiency. The formation of the lens pre-placode appears to be unaffected in heterozygotes, as deduced from the number of cells, the mitotic index, the amount of apoptosis and the expression of SOX2 and Pax6 in the pre-placode. However, the formation of the lens placode is delayed. The cells at the edge of the lens cup fail to express N-cadherin and undergo apoptosis and the lens fails to detach completely from the surface ectoderm. After formation, the lens, which has 50% of the cells found in wild-type embryos, grows at a rate that is indistinguishable from wild type. The possibility that monoallelic expression of Pax6 at the time of lens placode formation accounts for the 50% reduction in cell number has been ruled out by showing that expression of Pax6 is biallelic in the lens placode and optic vesicle. It is proposed instead that a critical threshold of PAX6 protein is required for lens placode formation and that the time in development at which this level is reached is delayed in heterozygotes (van Raamsdonk, 2000).

The molecular mechanisms mediating the retinogenic potential of multipotent retinal progenitor cells (RPCs) are poorly defined. Prior to initiating retinogenesis, RPCs express a limited set of transcription factors implicated in the evolutionary ancient genetic network that initiates eye development. The function of one of these factors, Pax6, in the RPCs of the intact developing eye, is elucidated by conditional gene targeting. Upon Pax6 inactivation, the potential of RPCs becomes entirely restricted to only one of the cell fates normally available to RPCs, resulting in the exclusive generation of amacrine interneurons. These findings demonstrate furthermore that Pax6 directly controls the transcriptional activation of retinogenic bHLH factors that bias subsets of RPCs toward the different retinal cell fates, thereby mediating the full retinogenic potential of RPCs (Marquardt, 2001).

By employing the Cre-loxP strategy to conditionally inactivate Pax6 in the lens primordium, it has been shown that Pax6 activity in the head surface ectoderm is autonomously required for lens formation. The complete absence of lens structures nevertheless permits the formation of a properly laminated retina containing all retinal cell types, indicating a striking independence of retinogenesis from the interaction with the developing lens. The developmental arrest in optic vesicle formation in the Pax6-/- mutant can therefore be attributed to an autonomous function of Pax6 during early optic vesicle genesis. Despite the continued expression of a number of factors that are indicative for early retinal development, the absence of Pax6 activity in the Pax6-/- optic vesicle leads to a failure in the transition from the neuroectoderm of the optic vesicle to the establishment of RPC identity. Consequently, neurogenesis, including the upregulation of neurogenic bHLH factors (NeuroD, Ngn2) and the appearance of betaIII-tubulin positive or syntaxin positive differentiating neurons, was not observed in the Pax6-/- optic vesicle rudiments. Additionally, the Pax6 alpha enhancer fails to be activated in Pax6-/- alpha-Cre optic vesicles. These results finally demonstrate that once RPC identity is established, Pax6 becomes dispensable for the maintenance of retinal identity. During the ensuing stages of retinogenesis, the continued presence of Pax6 activity eventually ensures the maintenance of the potential of RPCs to generate all retinal cell types (Marquardt, 2001).

During normal retinogenesis, Pax6 becomes downregulated in most cell lineages, but is maintained at a high level in all amacrine cells. The observation of wild-type-like amacrine cell genesis in the Pax6floxDelta (Pax6-) retina and the generation of terminally differentiated amacrine cells demonstrates the dispensability of Pax6 activity for the differentiation and maintenance of this cell type. A similar observation was made for the endocrine lineage of the developing pancreas, where Pax6 activity is present at a high level in both alpha and beta cells, but only the first fail to be generated in Pax6 null mutant mice. However, the observed near absence of glycinergic amacrine cells in the Pax6floxDelta retina indicates that Pax6 activity is indeed not dispensable for all amacrine cell classes (Marquardt, 2001).

In the wild-type situation, the onset of amacrine cell genesis is preceded by or coincides with the differentiation of retinal ganglion cells, cone photoreceptors, and horizontal cells, all of which fail to be generated in the Pax6floxDelta retina. Hence, Pax6 inactivation does not lead to the preferential generation of the only cell fate available by the time inactivation occurs. Furthermore, bipolar and Müller glia cells start to differentiate at E14 and E16, while in the Pax6floxDelta retina, the first differentiating (syntaxin postive and betaIII-tubulin positive) cells appear around E14. Only 1 day following birth (PN1), the majority of the cells in the Pax6floxDelta retina constitute differentiated amacrine cells, that is, 2 days before the peak, but 4-6 days after onset of Müller glia and bipolar cell differentiation. The gradual appearance of syntaxin positive cells, moreover, indicates that the inactivation of Pax6 does not lead to an immediate switch to a lineage-restricted (amacrine and horizontal cell generating, RPC population. The absence of late-born cell types in the Pax6floxDelta retina, therefore, cannot be attributed to a premature depletion of the Pax6floxDelta RPC pool. The normal timing of cell differentiation possibly reflects the largely unaffected Notch signaling in the Pax6floxDelta retina, as indicated by the initiation of Hes1, Notch1, and Delta-like1 expression (albeit the latter two at lower levels). Finally, the severely reduced RPC proliferation or premature cell cycle exit in Chx10 and Hes1 mutant mice, or the block of DNA synthesis in Xenopus embryos, all have little effect on the determination of retinal cell fate. Therefore, the observed restriction to the amacrine cell fate in the Pax6floxDelta retina cannot be indirectly attributed to a decreased level of RPC proliferation. The Pax6floxDelta RPCs rather appear to be intrinsically restricted in their retinogenic potential to only one of the different retinal cell fates normally available to RPCs (Marquardt, 2001).

The results indicate that the direct control of retinogenic bHLH factor expression constitutes a general mechanism of how Pax6 executes its function in retinal neurogenesis. bHLH transcription factors have been shown to play an essential role in the differentiation of retinal cell types from RPCs. However, no overt defect could so far be detected in the retina of Ngn2-deficient mice, which possibly is due to compensation by upregulation of Mash1, as has been previously demonstrated for the cerebral cortex. Preliminary results indicate that Ngn2; Mash1 double mutants display a complete absence of bipolar cells. Since Mash1 null mutants alone exhibit reduced (but not absent) bipolar cell differentiation, this observation suggests that during normal retinogenesis, bipolar cells evolve from two nonoverlapping RPC populations that are defined by the activity of Ngn2 and Mash1, which in turn require Pax6 for their expression (Marquardt, 2001).

The exclusive commitment of Pax6floxDelta RPCs to the amacrine cell fate is apparently due to the Pax6-independent activation of the amacrine cell differentiation program, for which the expression of NeuroD is indicative. Interestingly, in most areas of the developing CNS, NeuroD expression is observed to be dependent on the activity of Neurogenins. Since Ngn2 fails to be expressed in Pax6floxDelta RPCs, the continued expression of factors like Six3 appears to be sufficient to mediate transcriptional activation of NeuroD. Most interestingly, although only retinal ganglion cells fail to be generated in the retina of Math5-deficient mice, the abolishment of this Pax6-dependent cell differentiation pathway alone results in a similar, yet partial, shift toward the amacrine cell fate. Future studies will have to address whether this cell fate shift is similarly mediated by an upregulation of NeuroD in the RPCs that normally express Math5 (Marquardt, 2001).

The selection of subsets of RPCs to activate retinogenic bHLH transcription factors presumably involves the interplay of long-range extrinsic cues (like Shh- or EGF-mediated signaling) and short-range cellular interactions, as mediated by Notch-Delta signaling. However, for their subsequent transcriptional activation and thereby the progression from undetermined to lineage-restricted RPC, the presence of Pax6 activity in the RPCs appears to be strictly prerequisite. These factors then impose a bias on these subsets of RPCs, thereby leading to the transition from uncommitted RPC toward a lineage-restricted RPC intermediate. By directly controlling this transition process, Pax6 mediates the full retinogenic potential of RPCs and hence, their multi-potency (Marquardt, 2001).

In the developing retina of amphibia and fish, retinal cells are generated in the ciliary marginal zone (CMZ) in the peripheral retina and continue to be added throughout adult life. In higher vertebrates, the regenerative capacity of the adult retina has been lost. Recently, however, evidence has been provided that retinal stem cells can be retrieved from the pigmented ciliary body of the adult mammalian retina, possibly reflecting an evolutionary homology to the CMZ of lower vertebrates. The least determined RPCs (retinal stem cells) in the peripheral CMZ coexpress the transcription factors Pax6, Rx1, and Six3, prior to the activation of proneural genes and the subsequent steps toward retinal neurogenesis. In the mammalian retina, RPCs similarly express a limited set of transcription factors, Pax6, Rx1, Six3, Six6, and Lhx2, prior and during the ensuing steps of retinogenesis. Pax6, Rx1, Six3, and Six6 have been implicated to constitute key components of an evolutionary conserved genetic network that initiates eye development. Given their role in initiating the events that ultimately lead to retinal development, the characteristic expression profile of Pax6, Rx1, Six3, Six6, and Lhx2 indicate an additional requirement for the combined action of these factors in conferring and/or maintaining RPC identity. In this study, an essential role could be defined for Pax6, one member of this limited set of transcription factors, in mediating the retinogenic potential of RPCs, thereby strongly supporting this assumption. Pax6-deficient RPCs maintain retinal characteristics, including the expression of Rx1, Six3, Six6, and Lhx2, and undergo neuronal differentiation, although their potential has become restricted to one single cell fate. These observations strongly indicate that the continued functions of these other factors are sufficient to maintain retinal identity. Factors like Six3 might be essential to permit the generation of amacrine cells in the absence of Pax6 function. The challenge lies now in addressing the function of each of the other members of this class of factors in RPCs, like Rx1, Six3, Six6, and Lhx2, in mediating and maintaining the retinogenic potential of RPCs (Marquardt, 2001).

Pax6 is a key transcription factor in eye development, particularly in lens development, but its molecular action has not been clarified. Pax6 initiates lens development by forming a molecular complex with SOX2 (most closely related to Drosophila Sox Neuro) on the lens-specific enhancer elements, e.g., the delta-crystallin minimal enhancer DC5. DC5 shows a limited similarity to the binding consensus sequence of Pax6 and is bound poorly by Pax6 alone. However, Pax6 binds cooperatively with SOX2 to the DC5 sequence, resulting in formation of a high-mobility form of ternary complex in vitro, which correlates with the enhancer activation in vivo. Pax6 and SOX2-interdependent factor occupancy of DC5 is observed in a chromatin environment in vivo, providing the molecular basis of synergistic activation by Pax6 and SOX2. Subtle alterations of the Pax6-binding-site sequence of DC5 or of the inter-binding-sites distance diminish the cooperative binding and causes formation of a non-functional low-mobility form complex, suggesting DNA sequence-guided and protein interaction-induced conformation change of the Pax6 protein. When ectopically expressed in embryo ectoderm, Pax6 and SOX2 in combination activate delta-crystallin gene and elicit lens placode development, indicating that the complex of Pax6 and SOX2 formed on specific DNA sequences is the genetic switch for initiation of lens differentiation (Kamachi, 2001).

Embryonic development requires a complex series of relative cellular movements and shape changes that are generally referred to as morphogenesis. Although some of the mechanisms underlying morphogenesis have been identified, the process is still poorly understood. This study addresses mechanisms of epithelial morphogenesis using the vertebrate lens as a model system. The apical constriction of lens epithelial cells that accompanies invagination of the lens placode is dependent on cytoskeletal protein Shroom3, a molecule associated with apical constriction during morphogenesis of the neural plate. Shroom3 is required for the apical localization of F-actin and myosin II, both crucial components of the contractile complexes required for apical constriction, and for the apical localization of Vasp, a Mena family protein with F-actin anti-capping function that is also required for morphogenesis. Finally, the expression of Shroom3 is shown to be dependent on the crucial lens-induction transcription factor Pax6. This provides a previously missing link between lens-induction pathways and the morphogenesis machinery and partly explains the absence of lens morphogenesis in Pax6-deficient mutants (Plageman, 2010).

The physical contact of optic vesicle with head surface ectoderm is an initial event triggering eye morphogenesis. This interaction leads to lens specification followed by coordinated invagination of the lens placode and optic vesicle, resulting in formation of the lens, retina and retinal pigmented epithelium. Although the role of Pax6 in early lens development has been well documented, its role in optic vesicle neuroepithelium and early retinal progenitors is poorly understood. This study shows that conditional inactivation of Pax6 at distinct time points of mouse neuroretina development has a different impact on early eye morphogenesis. When Pax6 is eliminated in the retina at E10.5 using an mRx-Cre transgene, after a sufficient contact between the optic vesicle and surface ectoderm has occurred, the lens develops normally but the pool of retinal progenitor cells gradually fails to expand. Furthermore, a normal differentiation program is not initiated, leading to almost complete disappearance of the retina after birth. By contrast, when Pax6 was inactivated at the onset of contact between the optic vesicle and surface ectoderm in Pax6(Sey/flox) embryos, expression of lens-specific genes was not initiated and neither the lens nor the retina formed. These data show that Pax6 in the optic vesicle is important not only for proper retina development, but also for lens formation in a non-cell-autonomous manner (Klimova, 2014).

Mutations in mammalian Pax-6 homologs: Pax-6 role in neural patterning

During vertebrate development, the specification of distinct cell types is thought to be controlled by inductive signals acting at different concentration thresholds. The degree of receptor activation in response to these signals is a known determinant of cell fate, but the later steps at which graded signals are converted into all-or-none distinctions in cell identity remain poorly resolved. In the ventral neural tube, motor neuron and interneuron generation depends on the graded activity of the signaling protein Sonic hedgehog (Shh). These neuronal subtypes derive from distinct progenitor cell populations that express the homeodomain proteins Nkx2.2 or Pax6 in response to graded Shh signaling. In mice lacking Pax6, progenitor cells generate neurons characteristic of exposure to greater Shh activity. However, Nkx2.2 expression expands dosally in Pax6 mutants, raising the possibility that Pax6 controls neuronal pattern indirectly. Evidence that Nkx2.2 has a primary role in ventral neuronal patterning. In Nkx2.2 mutants, Pax6 expression is unchanged but cells undergo a ventral-to-dorsal transformation in fate and generate motor neurons rather than interneurons. Thus, Nkx2.2 has an essential role in interpreting graded Shh signals and selecting neuronal identity (Briscoe, 1999).

Distinct classes of motor neurons (MNs) and ventral interneurons are generated by the graded signaling activity of Sonic hedgehog (Drosophila homolog: Hedgehog). Three classes of ventral neurons are induced by distinct concentration thresholds of Shh. One class of interneurons, V1, is defined by coexpression of En1, Lim1/2 and Pax2 and is generated in the dorsal-most region of the ventral spinal cord. A second class of interneurons, V2, is defined by coexpression of Chx10, Lim3, and Gsh4 and is generated in the intermediate region of the ventral spinal cord, in a aregion that is ventral to V1 neurons. The third class, MNs, is defined by expression of Isl1 and is generated ventral to V2 interneurons. Shh controls neuronal fate by establishing different progenitor cell populations in the ventral neural tube that are defined by the expression of Pax6 and Nkx2.2 (a homolog of Drosophila Vnd). Pax6 establishes distinct ventral progenitor cell populations and controls the identity of motor neurons and ventral interneurons, mediating graded Shh signaling in the ventral spinal cord and hindbrain. From stages 10 to 12, Pax6 is expressed by cells at all dorsoventral positions of the neural tube, with the exception of the ventral midline. Nkx2.2 is also detected at low levels but is restricted to ventral midline cells. From stages 12 to 16, the level of Pax 6 in cells adjacent to the floor plate decreases below the limit of detection; Nkx2.2 expression is initiated within these cells. Both V1 and V2 neurons derive from Pax 6 precursor cells, while ventrally positioned MNs derive from Nkx2.2 progenitor cells. In Pax6 mutant mice there is a marked increase in the number of Nkx2.2 cells and a dorsal expansion of the Nkx2.2 domain. Loss of Pax6 results in a dorsal-to-ventral transformation of one type of MNs to another (Ericson, 1997).

In mammals, Pax-6 is expressed in several discrete domains of the developing CNS and has been implicated in neural development, although its precise role remains elusive. A novel Small eye rat strain (rSey2) was found with phenotypes similar to mouse and rat Small eye. Analyses of the Pax-6 gene reveals one base (C) insertion in an exon encoding the region downstream of the paired box of the Pax-6 gene (resulting in the rSey2 mutation), resulting in the generation of a truncated protein due to the frame shift. rSey2/rSey2 mutant rats exhibit abnormal development of motor neurons in the hindbrain. The Islet-1-positive motor neurons are generated just ventral to the Pax-6-expressing domain, both in the wild-type and mutant embryos. However, two somatic motor (SM) nerves, the abducens and hypoglossal nerves, are missing in homozygous embryos. No SM-type axonogenesis (ventrally growing) is found in the mutant postotic hindbrain, though branchiomotor and visceral motor (BM/VM)-type axons (dorsally growing) are observed within the neural tube. To discover whether the identity of these motor neuron subtypes is changed in the mutant, expression of LIM homeobox genes Islet-1, Islet-2 (See Drosophila Islet)and Lim-3 were examined. At the postotic levels of the hindbrain, SM neurons express all the three LIM genes, whereas BM/VM-type neurons are marked by Islet-1 only. In the Pax-6 mutant hindbrain, Islet-2 expression is specifically missing, which results in the loss of the cells harboring the post-otic hindbrain SM-type LIM code (Islet-1 + Islet-2 + Lim-3). Expression of Wnt-7b, which overlaps with Pax-6 in the ventrolateral domain of the neural tube, is also specifically missing in the mutant hindbrain, while it remained intact in the dorsal non-overlapping domain. These results strongly suggest that Pax-6 is involved in the specification of subtypes of hindbrain motor neurons, presumably through the regulation of Islet-2 and Wnt-7b expression. Since Islet-2 and Pax-6 expression domains do not overlap, is suggested that Islet-2 expression is regulated by indirecly by Pax-6 via Wnt-7b. This makes evolutionary sense as it is known that Drosophila paired regulates wingless and Wnt-1 expression in the midbrain/hindbrain boundary is controlled by Pax-2,5, 8. Wnt-7b may be involved in either specification of neuronal subdypes or axon pathfinding (Osumi, 1997).

Different neuronal subpopulations derived from in vitro differentiation of embryonic stem (ES) cells have been characterized using as markers the expression of several homeodomain transcription factors. Following treatment of embryo-like aggregates with retinoic acid (RA), Pax-6, a protein expressed by ventral central nervous system (CNS) progenitors, is induced. In contrast, Pax-7 expressed in vivo by dorsal CNS progenitors, and erbB3, a gene expressed by neural crest cells and its derivatives, are almost undetectable. CNS neuronal subpopulations generate expressed combination of markers characteristic of somatic motoneurons (Islet-1/2, Lim-3, and HB-9); cranial motoneurons (Islet-1/2 and Phox2b) and interneurons (Lim-1/2 or EN1). Molecular characterization of neuron subtypes generated from ES cells should considerably facilitate identification of new genes expressed by restricted neuronal cell lineages (Renoncourt, 1998).

The nested expression patterns of the paired-box containing transcription factors Pax2/5 and Pax6 demarcate the midbrain and forebrain primordium at the neural plate stage. In Pax2/5 deficient mice, the mesencephalon/metencephalon primordium is completely missing, resulting in a fusion of the forebrain to the hindbrain. Morphologically, in the alar plate the deletion is characterized by the substitution of the tectum (dorsal midbrain) and cerebellum (dorsal metencephalon) by the caudal diencephalon and in the basal plate by the replacement of the midbrain tegmentum by the ventral metencephalon (pons). Molecularly, the loss of the tectum is demonstrated by an expanded expression of Pax6, (the molecular determinant of posterior commissure), and a rostral shift of the territory of expression of Gbx2 and Otp (markers for the pons), toward the caudal diencephalon. These results suggest that an intact territory of expression of Pax2/5 in the neural plate, nested between the rostral and caudal territories of expression of Pax6, is necessary for defining the midbrain vesicle (Schwarz, 1999).

It is not clear to what extent restricted cell migration contributes to patterning of the developing telencephalon, since both restricted and widespread cell migration have been observed. Dorso-ventral cell migration in the telencephalon of Pax6 mutant mice (Small Eye) has been examined. The transcription factor Pax6 is expressed in the dorsal telencephalon, the cerebral cortex. Focal injections of adenoviral vectors containing the green fluorescent protein were used to follow and quantify cell movements between two adjacent regions in the developing telencephalon, the cerebral cortex and the ganglionic eminence (the prospective basal ganglia). The analysis in wild-type mice confirmed that the cortico-striatal boundary acts as a semipermeable filter and allows a proportion of cells from the ganglionic eminence to invade the cortex, but not vice versa. Ventro-dorsal cell migration is strongly enhanced in the Pax6 mutant. An essential function of Pax6 in the regionalization of the telencephalon is then to limit the invasion of the cortex by cells originating in the ganglionic eminence. Cortical cells, however, remain confined to the cortex in the Pax6 mutant. Thus, dorsal and ventral cells are restricted to their respective territories by distinct mechanisms (Chapouton, 1999).

Which mechanisms could then be responsible for the migrational restriction of cells from the GE? Since the migration of GE cells into the cortex is free in the absence of functional Pax6, the cues that restrict GE cells should be lost in this mutant. Several changes have been detected in the Pax6 mutant with regard to the delineation between the cortex and GE. The radial glia fascicle at the cortico-striatal boundary region and also the cortex-specific expression of R-cadherin are both absent in the Pax6 mutant Sey. Thus, the absence of the mechanical obstacle formed by the radial glia fascicle may ease migration from the GE. Alternatively, the loss of R-cadherin on cortical cells may permit the intermingling of GE cells with cortical cells that is less favored in the wild type. Needless to say, other as yet uncharacterized molecules may well contribute to the specific migrational restriction of GE cells. The Pax6 mutant should serve as an excellent model for investigating which molecules co-operate at the cortico-striatal boundary to restrict the migration of ventral cells (Chapouton, 1999).

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. Pax6 was misexpressed 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 indicate that Pax6 misexpression causes a caudal shift of the di-mesencephalic boundary. Pax6 represses En1, Pax2 and other tectum (mesencephalon)-related genes such as En2, Pax5, Pax7, but induces Tcf4, a diencephalon marker gene. To know how Pax6 represses En1 and Pax2, a dominant-active or negative form of Pax6 was ectopically expressed. The dominant-active form of Pax6 shows a similar but more severe phenotype than Pax6, while the dominant-negative form shows 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 (Matsunaga, 2000).

Recent studies have shown that the generation of different kinds of neurons is controlled by combinatorial actions of homeodomain (HD) proteins expressed in the neuronal progenitors. Pax6 is a HD protein that is involved in the differentiation of the hindbrain somatic (SM) motoneurons and V1 interneurons in the hindbrain and/or spinal cord. To investigate in greater depth the role of Pax6 in generation of the ventral neurons, the expression patterns were examined of HD protein genes and subtype-specific neuronal markers in the hindbrain of the Pax6 homozygous mutant rat. Islet2 (SM neuron marker) and En1 (V1 interneuron marker) are transiently expressed in a small number of cells, indicating that Pax6 is not directly required for specification of these neurons. Domains of all other HD protein genes (Nkx2.2, Nkx6.1, Irx3, Dbx2 and Dbx1) were shifted and their boundaries became blurred. Thus, Pax6 is required for establishment of the progenitor domains of the ventral neurons. Next, Pax6 overexpression experiments were performed by electroporating rat embryos in whole embryo culture. Pax6 overexpression in the wild type decreases expression of Nkx2.2, but ectopically increases expression of Irx3, Dbx1 and Dbx2. Moreover, electroporation of Pax6 into the Pax6 mutant hindbrain rescues the development of Islet2-positive and En1-positive neurons. To know reasons for perturbed progenitor domain formation in Pax6 mutant, expression patterns of Shh signaling molecules and states of cell death and cell proliferation were examined. Shh is similarly expressed in the floor plate of the mutant hindbrain, while the expressions of Ptc1, Gli1 and Gli2 are altered only in the progenitor domains for the motoneurons. The position and number of TUNEL-positive cells are unchanged in the Pax6 mutant. Although the proportion of cells that are BrdU-positive slightly increases in the mutant, there is no relationship with specific progenitor domains. Taken together, it is concluded that Pax6 regulates specification of the ventral neuron subtypes by establishing the correct progenitor domains (Takahashi, 2002).

The most important finding in this study is that loss of Pax6 function leads to failure in formation of the correct progenitor domains within the ventricular zone. The expression boundaries of all HD protein genes are blurred and shifted in the Pax6 mutant. Expression of Nkx2.2 and Dbx2 expands dorsally, while that of Nkx6.1, Irx3 and Dbx1 shifts ventrally. The altered expression patterns of the HD code genes in the Pax6 mutant explain very well why a small number of V1 interneurons and SM neurons emerge; the progenitor domains for V1 interneurons and SM neurons, which are defined by the expression boundaries of Dbx1/Dbx2 and Irx3/Nkx2.2, respectively, are formed as extremely narrow domains in the Pax6 mutant. Emergence of these V1 and SM neurons may be transient (only for ~10 hours) because these expression boundaries are not firmly maintained and such neurons will be diminished soon after. By contrast, progenitor domains for BM neurons, V2 interneurons and V0 interneurons become expanded. This is consistent with the observation that the number of V2 interneurons increases in the mutant rat (Takahashi, 2002).

If Pax6 is required for establishment of the progenitor domains in a correct manner, is it sufficient for progenitor domain formation? To answer this question, overexpression of Pax6 by electroporation into cultured rat embryos was performed, and indeed development of SM neurons and V1 interneurons were rescued in correct positions. The numbers of these rescued neurons were less than in normal development. The reason for this partial rescue may be that exogenous Pax6 cannot fully re-establish the progenitor domains for SM neurons and V1 interneurons at the stage of electroporation. Irx3, the gene reported to repress SM fate in the chick spinal cord, is already expanded ventrally at the time of electroporation. Alternatively, exogenous Pax6 could not cause a complete repression of the expanded expression of Nkx2.2. Taking these loss-of-function and gain-of-function studies together, it is concluded that Pax6 seems to regulate formation of the precursor domains in the hindbrain, thereby specify the fates of ventral neurons (Takahashi, 2002).

During brain development, Pax6 is expressed in specific regions of the diencephalon including secretory cells of the subcommissural organ (SCO), a circumventricular organ at the forebrain-midbrain boundary that originates from the pretectal dorsal midline neuroepithelial cells beneath the posterior commissure (PC). Homozygous small eye (Sey/Sey) mice lack functional Pax6 protein and fail to develop the SCO, a normal PC and the pineal gland. Small eye heterozygotes (Sey/+) show defective development of the SCO's basal processes that normally penetrate the PC, indicating that normal development of the gland requires normal Pax6 gene-dosage. A correlation between the defects of SCO formation and altered R- and OB-cadherin expression patterns in the SCO is observed in mutants, suggesting a role for cadherins in SCO development (Estivill-Torrus, 2001).

Levels of expression of the transcription factor Pax6 vary throughout corticogenesis in a rostro-lateralhigh to caudo-mediallow gradient across the cortical proliferative zone. Previous loss-of-function studies have indicated that Pax6 is required for normal cortical progenitor proliferation, neuronal differentiation, cortical lamination and cortical arealization, but whether and how its level of expression affects its function is unclear. The developing cortex was studied of PAX77 YAC transgenic mice carrying several copies of the human PAX6 locus with its full complement of regulatory regions. It was found that PAX77 embryos express Pax6 in a normal spatial pattern, with levels up to three times higher than wild type. By crossing PAX77 mice with a new YAC transgenic line that reports Pax6 expression (DTy54), it was shown that increased expression is limited by negative autoregulation. Increased expression reduces proliferation of late cortical progenitors specifically, and analysis of PAX77 --> wild-type chimeras indicates that the defect is cell autonomous. Cortical arealization was studied in PAX77 mice and it was found that, whereas the loss of Pax6 shifts caudal cortical areas rostrally, Pax6 overexpression at levels predicted to shift rostral areas caudally has very little effect. These findings indicate that Pax6 levels are stabilized by autoregulation, that the proliferation of cortical progenitors is sensitive to altered Pax6 levels and that cortical arealization is not (Manuel, 2007).

Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. Pax6 was used as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. The genomic binding locations were identified of Pax6 in neocortical stem cells during normal development, and the functional significance of genes were ascertained that were found to be regulated by Pax6. Pax6 was found to positively and directly regulate cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, a core network regulating neocortical stem cell decision-making was identified in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. It was found that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal (Sansom, 2009).

Cell-autonomous repression of Shh by transcription factor Pax6 regulates diencephalic patterning by controlling the central diencephalic organizer.

During development, region-specific patterns of regulatory gene expression are controlled by signaling centers that release morphogens providing positional information to surrounding cells. Regulation of signaling centers themselves is therefore critical. The size and the influence of a Shh-producing forebrain organizer (see Drosophila Hedgehog), the zona limitans intrathalamica (ZLI), are limited by Pax6. By studying mouse chimeras, this study found that Pax6 acts cell autonomously to block Shh expression in cells around the ZLI. Immunoprecipitation and luciferase assays indicate that Pax6 can bind the Shh promoter and repress its function. An analysis of chimeras suggests that many of the regional gene expression pattern defects that occur in Pax6-/- diencephalic cells result from a non-cell-autonomous position-dependent defect of local intercellular signaling. Blocking Shh signaling in Pax6-/- mutants reverses major diencephalic patterning defects. It is concluded that Pax6's cell-autonomous repression of Shh expression around the ZLI is critical for many aspects of normal diencephalic patterning (Caballero, 2014: PubMed).

Mutations in mammalian Pax-6 homologs: Effects on cell migration

The olfactory bulbs are structures that protrude from the rostral telencephalon and function as the primary processing center for odor information. The OB has a laminar structure, consisting of several types of neurons and glial cells. Mitral cells, which differentiate first among the OB neurons, receive direct axonal innervation from olfactory receptor neurons in the olfactory epithelium and their axons project to the olfactory cortex. Several interneurons also have important roles in local circuits within the OB. However, it is unclear how the OB forms at the precise position of the rostral telencephalon. Pax6-mutant mice and rats lack the olfactory bulb and, instead, develop an olfactory bulb-like structure at the lateral part of the telencephalon. Ectopic formation of the olfactory bulb-like structure in these mutants is caused by the abnormal migration of mitral cell progenitors, which first differentiate within the olfactory bulb. Cell-tracing experiments in whole embryos in culture indicate that, in the mutants, the mitral cell progenitors that originate from the rostral part of the telencephalon migrate caudally toward the lateral part of the telencephalon. Cell transplantation demonstrates that the abnormal cell migration is not autonomous to the mitral cell progenitors themselves. The mislocation of the olfactory bulb in the mutant is not caused by loss of olfactory nerve innervation. Furthermore, transfection of a Pax6-expression vector to the mutant telencephalon restores the normal migration of mitral cell progenitors. These results provide evidence that Pax6 is required to position the mitral cell progenitors at the rostral end of the telencephalon (Nomura, 2004).

Mutations in mammalian Pax-6 homologs: Pax-6 role in pancreas development

PAX6 is a key regulator of pancreatic islet hormone gene transcription and is required for normal islet development. In embryos homozygous for a mutant allele of the Pax6 gene, the numbers of all four types of endocrine cells in the pancreas are decreased significantly, and islet morphology is abnormal. In the remaining islet cells, hormone production, particularly glucagon production, is markedly reduced because of decreased gene transcription. These effects appear to result from a lack of PAX6 protein in the mutant embryos. Biochemical studies identify wild-type PAX6 protein as the transcription factor that binds to a similar element in the insulin, glucagon, and somatostatin promoters. These studies show that PAX6 transactivates the glucagon and insulin promoters (Sander, 1997).

The specification of the different mouse pancreatic endocrine subtypes is determined by the concerted activities of transcription factors. However, the molecular mechanisms regulating endocrine fate allocation remain unclear. In the present study, the molecular consequences were uncovered of the simultaneous depletion of Arx and Pax4 activity during pancreas development. The findings reveal a so far unrecognized essential role of the paired-box-encoding Pax4 gene. Specifically, in the combined absence of Arx and Pax4, an early-onset loss of mature alpha- and ß-cells occurs in the endocrine pancreas, concomitantly with a virtually exclusive generation of somatostatin-producing cells. Furthermore, despite normal development of the PP-cells in the double-mutant embryos, an atypical expression of the pancreatic polypeptide (PP) hormone was observed in somatostatin-labelled cells after birth. Additional characterizations indicate that such an expression of PP is related to the onset of feeding, thereby unravelling an epigenetic control. Finally, the data provide evidence that both Arx and Pax4 act as transcriptional repressors that control one another's expression levels, thereby mediating proper endocrine fate allocation (Collombat, 2005).

Canonical Notch signaling is thought to control the endocrine/exocrine decision in early pancreatic progenitors. Later, RBP-Jkappa interacts with Ptf1a and E12 to promote acinar differentiation. To examine the involvement of Notch signaling in selecting specific endocrine lineages, this pathway was deregulated by targeted deletion of presenilin1 and presenilin2, the catalytic core of gamma-secretase, in Ngn3- or Pax6-expressing endocrine progenitors. Surprisingly, whereas Pax6(+) progenitors were irreversibly committed to the endocrine fate, it was discovered that Ngn3(+) progenitors were bipotential in vivo and in vitro. When presenilin amounts are limiting, Ngn3(+) progenitors default to an acinar fate; subsequently, they expand rapidly to form the bulk of the exocrine pancreas. gamma-Secretase inhibitors confirmed that enzymatic activity was required to block acinar fate selection by Ngn3 progenitors. Genetic interactions identified Notch2 as the substrate, and suggest that gamma-secretase and Notch2 act in a noncanonical titration mechanism to sequester RBP-Jkappa away from Ptf1a, thus securing selection of the endocrine fate by Ngn3 progenitors. These results revise the current view of pancreatic cell fate hierarchy, establish that Ngn3 is not in itself sufficient to commit cells to the endocrine fate in the presence of Ptf1a, reveal a noncanonical action for Notch2 protein in endocrine cell fate selection, and demonstrate that acquisition of an endocrine fate by Ngn3(+) progenitors is gamma-secretase-dependent until Pax6 expression begins (Cras-Méneur, 2009).

Mutations in mammalian Pax-6 homologs: Pax-6 role development of other glands

The mechanisms behind the cell-specific and compartmentalized expression of gut and pancreatic hormones is largely unknown. Deletion of the Pax 4 gene virtually eliminates duodenal and jejunal hormone-secreting cells, as well as serotonin and somatostatin cells of the distal stomach, while deletion of the Pax 6 gene eliminates duodenal GIP cells as well as gastrin and somatostatin cells of the distal stomach. Thus, together, these two genes regulate the differentiation of all proximal gastrointestinal endocrine cells and reflect common pathways for pancreatic and gastrointestinal endocrine cell differentiation (Larsson, 1998).

The mechanism of tissue induction and specification was examined using the lacrimal gland as a model system. This structure begins its morphogenesis as a bud-like outgrowth of the conjunctival epithelium and ultimately forms a branched structure with secretory function. Using a reporter transgene as a specific marker for gland epithelium, it has been shown that the transcription factor Pax6 is required for normal development of the gland and is probably an important competence factor. In investigating the cell-cell signaling required, it has been shown that FGF10 is sufficient to stimulate ectopic lacrimal bud formation in ocular explants. Expression of FGF10 in the mesenchyme adjacent to the presumptive lacrimal bud and absence of lacrimal gland development FGF10-null mice strongly suggest that it is an endogenous inducer. This was supported by the observation that inhibition of signaling by a receptor for FGF10 (receptor 2 IIIb) suppressed development of the endogenous lacrimal bud. In explants of mesenchyme-free gland epithelium, FGF10 stimulates growth but not branching morphogenesis. This suggests that its role in induction is to stimulate proliferation and, in turn, that FGF10 combines with other factors to provide the instructive signals required for lacrimal gland development. The lack of normal lacrimal gland formation in the Sey heterozygous mice indicates that the Pax6 transcription factor is required. Pax6 is expressed in the conjunctival epithelium but not in the neural crest-derived periocular mesenchyme. This suggests that the requirement for Pax6 in gland formation is autonomous to the cells of the precursor conjunctival epithelium and is consistent with an autonomous requirement for Pax6 in the formation of the lens. It is likely that Pax6 is one factor that establishes competence and permits gland development from conjunctival epithelium in response to an FGF ligand (Makarenkova, 2000).

Table of contents

eyeless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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