eyeless

EVOLUTIONARY HOMOLOGS

Table of contents

Regulation of Pax-6 in mammals

Pax6 plays a key role in visual system development throughout the metazoa and the function of Pax6 is evolutionarily conserved. However, the regulation of Pax6 expression during eye development is largely unknown. Two physically distinct promoters in mouse Pax6, P0 and P1, have been identified that direct differential Pax6 expression in the developing eye. The two promoters are each associated with different 5' UTRs. P0-initiated transcripts predominate in lens placode and corneal and conjunctival epithelia, whereas P1-initiated transcripts are expressed in lens placode, optic vesicle and CNS, and only weakly in corneal and conjunctival epithelia. Several distinct Pax6 cDNAs containing either exon 0 or exon 1 at their 5'-ends were isolated. To determine whether the corresponding transcripts initiate from different promoters, their transcription start sites were determined by RNase protection and primer extension. RNase protection with exon 0- or exon 1-specific riboprobes yields a 154 bp protected fragment for exon 0 and several protected fragments for exon 1, the longest being 230 bp. The assignment of initiation sites was confirmed by primer extension. An antisense oligonucleotide complementary to either exon 0 or exon 1 results in 154 nt or 201 nt extension products respectively. These results indicate that exon 0 and exon 1 transcripts are initiated from two distinct promoters, P0 and P1. The initiation of transcripts containing exon 0 is at an A, 30 bp downstream of the putative P0 TATA box, while that of transcripts containing exon 1 is at a G, 23 bp downstream of the putative P1 TATA box. Moreover, the P0 and P1 promoter regions function as promoters in cultured cells (Xu, 1999).

To study whether the mouse P0 and P1 Pax6 promoters direct differential Pax6 expression during eye development, in situ hybridization was performed with probes specific to either P0- or P1-initiated transcripts, as well as with a third probe located in the 3'-UTR region that detects all Pax6 transcripts. P0 transcripts are observed abundantly in the lens placode at E9.5. In contrast, P1 transcripts are abundant in both the lens placode and optic vesicle. Consistent with these results, a Pax6 3'-UTR probe detects expression in both the lens placode and optic vesicle, with stronger expression in the lens placode. After E9.5, P0 transcripts increase in the retina, and by E13.5 become comparable in abundance and spatial distribution to P1 transcripts in lens and retina. At E17.5, both P0 and P1 transcripts are strongly expressed in lens epithelial cells and developing neuroretina, but P0 transcripts are more abundant in the inner layer, while P1 transcripts are distributed uniformly. Lastly, in the developing corneal and conjunctival epithelia, P0 transcripts are more abundant than P1 transcripts, and both transcripts are also differentially distributed in CNS development. The differential and overlapping expression of the two Pax6 promoters suggests that their expression is controlled by both promoter specific regulatory elements, and by elements capable of interacting with both promoters with different efficiencies (Xu, 1999).

It is concluded that the regulatory element upstream of the Pax6 P0 promoter is required for expression in a subpopulation of retinal progenitors and in the developing pancreas, while a second regulatory element upstream of the Pax6 P1 promoter is sufficient to direct expression in a subset of post-mitotic, non-terminally differentiated photoreceptors. An evolutionarily conserved 341 bp mouse Pax6 regulatory element has been identified that controls lens placode and corneal ectoderm expression (Williams, 1998). This enhancer resides between 3.9 and 3.5 kb upstream of the P0 promoter, just upstream of the longest 3.3 kb P0 construct examined in the current study. The identification of a Pax6 lens placode enhancer upstream of the P0 promoter is consistent with the results presented here demonstrating that P0-initiated transcripts are preferentially expressed in lens placode and corneal epithelium. A third element in Pax6 intron 4, when combined with either the P0 or P1 promoter, accurately directs expression in amacrine cells, ciliary body and iris. These results indicate that the complex expression pattern of Pax6 is differentially regulated by two promoters acting in combination with multiple cis-acting elements. Whether the regulatory mechanisms that direct Pax6 ocular expression are conserved between mice and flies was also tested. Remarkably, an eye-enhancer region of the Drosophila eyeless gene, when inserted upstream of either the mouse Pax6 P1 or P0 promoter, directs eye- and CNS-specific expression in transgenic mice that accurately reproduces features of endogenous Pax6 expression. These results suggest that in addition to conservation of Pax6 function, the upstream regulation of Pax6 has also been conserved during evolution (Xu, 1999).

The P1 regulatory element resides between 3.1 and 2.9 kb upstream of the P1 promoter. This element directs lacZ expression in postmitotic retinal cells that migrate to the outermost aspect of the retinal outer layer, coinciding temporally and spatially with cone cell genesis between E11 and E18. Although cone cells constitute only about 3% of the total photoreceptor cell population and the P1 transgene is expressed in a majority of outer layer cells at E13.5, this could reflect a maximal level of cone differentiation prior to the onset of rod differentiation, which mainly occurs postnatally. Consistent with the fact that Pax6 is not expressed in differentiated photoreceptors, lacZ expression was not observed after birth when most rod photoreceptors are born; nor is transgene expression detected in other retinal cell types. Furthermore, the P1-lacZ transgene expression correlates with the early expression pattern of a cone and rod specific homeobox gene Crx. Thus, while conclusive proof requires cell lineage analyses, the data suggest that the P1-lacZ transgene expressing cells are likely to be differentiating cone cells. Interestingly, a potentially analogous, post-mitotic pre-rod stage of rod photoreceptor differentiation has been identified in vitro. PAX6 overexpression in transgenic mice has been shown to result in an absence of photoreceptors. Pax6 may thus participate in photoreceptor differentiation, and the P1 element might regulate this function (Xu, 1999).

Although the eyeless regulatory elements appear to function in mice with considerable fidelity, the results from reciprocal experiments analyzing mouse Pax6 elements in Drosophila were not as clear. A mouse fragment containing the Pax6 P1 element is able to direct lacZ transgene expression in the Drosophila eye imaginal disc. This expression is restricted specifically to differentiating photoreceptors posterior to the morphogenetic furrow, which is not a site of endogenous Ey expression. Curiously, the eyeless transgenes also exhibit anomalous expression posterior to the furrow. The basis for the ey transgenic results is unclear, but could reflect beta-galactosidase perdurance or the absence of a repressive function from the ey transgene. In the case of the mouse P1 element, it is interesting that the cells that activate the P1 element in both the Drosophila eye imaginal disc and the mouse retina are developing photoreceptors. In fact, in the Drosophila adult eye and in Bolwig’s organ, a component of the larval visual system, ey is expressed in photoreceptors. Thus, although the expression of the mouse Pax6 P1 upstream fragment in Drosophila does not reproduce endogenous ey expression, the mechanisms regulating photoreceptor differentiation in mice and flies may still be conserved (Xu, 1999).

Pax6 is a regulatory gene with restricted expression and essential functions in the developing eye and pancreas and distinct domains of the CNS. Three conserved transcription start sites (P0, P1, alpha) have been identified in the murine Pax6 locus. Furthermore, using transgenic mouse technology independent cis-regulatory elements controlling the tissue-specific expression of Pax6 have been localized. Specifically, a 107-bp enhancer and a 1.1-kb sequence within the 4.6-kb untranslated region upstream of exon 0 are required to mediate Pax6 expression in the lens, cornea, lacrimal gland, conjunctiva, or pancreas, respectively. Another 530-bp enhancer fragment located downstream of the Pax6 translational start site is required for expression in the neural retina, the pigment layer of the retina, and the iris. Finally, a 5-kb fragment located between the promoters P0 and P1 can mediate expression into the dorsal telencephalon, the hindbrain, and the spinal cord. The identified Pax6/cis-essential elements are highly conserved in pufferfish, mouse, and human DNA and contain binding sites for several transcription factors indicative of the cascade of control events. Corresponding regulatory elements from pufferfish are able to mimic the reporter expression in transgenic mice. Thus, the results indicate a structural and functional conservation of the Pax6 regulatory elements in the vertebrate genome (Kammandel, 1999).

The function of Lhx2, a LIM homeobox gene expressed in developing B-cells, forebrain and neural retina, was analyzed by using embryos deficient in functional Lhx2 protein. Lhx2 mutant embryos are anophthalmic, have malformations of the cerebral cortex, and die in utero due to severe anemia. In Lhx2-/- embryos specification of the optic vesicle occurs, however, development of the eye arrests prior to formation of an optic cup. Pax-6 expression in the optic vesicle is normal in knockout mice, but Pax-6 expression in the ectoderm overlying the optic vesicle is deficient, suggesting the Lhx2 function in the optic vesicle is necessary for either induction or maintence of Pax6 expression in the presumptive lens ectoderm. Deficient cellular proliferation in the forebrain results in hypoplasia of the neocortex and aplasia of the hippocampal anlagen. In addition to the central nervous system malformations, a cell non-autonomous defect of definitive erythropoiesis causes severe anemia in Lhx2-/- embryos. It is thought that the cell non-autonomous defect is due to a defect in the fetal hepatic microenvironment. Thus Lhx2 is necessary for normal development of the eye, cerebral cortex, and efficient definitive erythropoiesis (Porter, 1997).

The transcription factor Pax6 is required for normal development of the central nervous system, eyes, nose, and pancreas. The transactivation domain (TAD) of zebrafish Pax6 is phosphorylated in vitro by the mitogen-activated protein kinases (MAPKs) extracellular-signal regulated kinase (ERK) and p38 kinase, but not by Jun N-terminal kinase (JNK). Three of four putative proline-dependent kinase phosphorylation sites are phosphorylated in vitro. Of these sites, the serine 413 (Ser413) is evolutionary conserved from sea urchin to man. Ser413 is also phosphorylated in vivo upon activation of ERK or p38 kinase. Substitution of Ser413 with alanine strongly decreases the transactivation potential of the Pax6 TAD, whereas substitution with glutamate increases the transactivation. Reporter gene assays with wild-type and mutant Pax6 reveal that transactivation by the full-length Pax6 protein from paired domain-binding sites is strongly enhanced (16-fold) following co-transfection with activated p38 kinase (see Drosophila p38b). This enhancement is largely dependent on the Ser413 site. ERK activation, however, produces a 3-fold increase in transactivation, which is partly independent of the Ser413 site. These findings provide a starting point for further studies aimed at elucidating a post-translational regulation of Pax6 following activation of MAPK signaling pathways (Mikkola, 1999).

Pax6 activation occurs in phase with somitogenesis in the spinal cord. The presomitic mesoderm exerts an inhibitory activity on Pax6 expression. This repressive effect is mediated by the FGF signaling pathway. The presomitic mesoderm displays a decreasing caudorostral gradient of FGF8, and grafting FGF8-soaked beads at the level of the neural tube abolishes Pax6 activation. Conversely, when FGF signaling is disrupted, Pax6 is prematurely activated in the neural plate. It is proposed that the progression of Pax6 activation in the neural tube is controlled by the caudal regression of the anterior limit of FGF activity. Hence, as part of its posteriorizing activity, FGF8 downregulation acts as a switch from early (posterior) to a later (anterior) state of neural epithelial development (Bertrand, 2000).

Fgf8, which is expressed at the embryonic mid/hindbrain junction, is required for and sufficient to induce the formation of midbrain and cerebellar structures. To address the genetic pathways through which FGF8 acts, the epistatic relationships of mid/hindbrain genes that respond to FGF8 were examined, using a novel mouse brain explant culture system. En2 and Gbx2 are the first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain explants treated with FGF8-soaked beads. By examining gene expression in En1/2 double mutant mouse embryos, it was found that Fgf8, Wnt1 and Pax5 do not require the En genes for initiation of expression, but do for their maintenance, and Pax6 expression is expanded caudally into the midbrain in the absence of EN function. Since E9.5 En1/2 double mutants lack the mid/hindbrain region, forebrain mutant explants were treated with FGF8 and, significantly, the EN transcription factors were found to be required for induction of Pax5. Thus, FGF8-regulated expression of Pax5 is dependent on EN proteins, and a factor other than FGF8 could be involved in initiating normal Pax5 expression in the mesencephalon/metencephalon. The En genes also play an important, but not absolute, role in repression of Pax6 in forebrain explants by FGF8. Gbx2 gain-of-function studies have shown that misexpression of Gbx2 in the midbrain can lead to repression of Otx2. However, in the absence of Gbx2, FGF8 can nevertheless repress Otx2 expression in midbrain explants. In contrast, Wnt1 is initially broadly induced in Gbx2 mutant explants, as in wild-type explants, but not subsequently repressed in cells near FGF8 that normally express Gbx2. Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts downstream of FGF8 in repression of Wnt1. This is the first such epistatic study performed in mouse that combines gain-of-function and loss-of-function approaches to reveal aspects of mouse gene regulation in the mesencephalon/metencephalon that have been difficult to address using either approach alone (Liu, 2001). The Pax6 gene has a central role in development of the eye. Through targeted deletion in the mouse, an ectoderm enhancer in the Pax6 gene has been shown to be required for normal lens formation. Ectoderm enhancer-deficient embryos exhibit distinctive defects at every stage of lens development. These include a thinner lens placode, reduced placodal cell proliferation, and a small lens pit and lens vesicle. In addition, the lens vesicle fails to separate from the surface ectoderm and the maturing lens is smaller and shows a delay in fiber cell differentiation. Interestingly, deletion of the ectoderm enhancer does not eliminate Pax6 production in the lens placode but results in a diminished level that, in central sections, is apparent primarily on the nasal side. This argues that Pax6 expression in the lens placode is controlled by the ectoderm enhancer and at least one other transcriptional control element. It also suggests that Pax6 enhancers active in the lens placode drive expression in distinct subdomains, an assertion that is supported by the expression pattern of a lacZ reporter transgene driven by the ectoderm enhancer. Interestingly, deletion of the ectoderm enhancer causes loss of expression of Foxe3, a transcription factor gene mutated in the dysgenetic lens mouse. This work allows the assembly of a more complete genetic pathway describing lens induction. This pathway features (1) a pre-placodal phase of Pax6 expression that is required for the activity of multiple, downstream Pax6 enhancers; (2) a later, placodal phase of Pax6 expression regulated by multiple enhancers; and (3) the Foxe3 gene in a downstream position. This pathway forms a basis for future analysis of lens induction mechanism (Dimanlig, 2001).

Fibroblast growth factor receptor (Fgfr) signaling plays a role in lens induction. Three distinct experimental strategies were used: (1) using small-molecule inhibitors of Fgfr kinase activity, it has been shown that both the transcription level and protein expression of Pax6, a transcription factor critical for lens development, is diminished in the presumptive lens ectoderm; (2) transgenic mice (designated Tfr7) that express a dominant-negative Fgf receptor exclusively in the presumptive lens ectoderm show defects in formation of the lens placode at E9.5 but in addition, showe reduced levels of expression for Pax6, Sox2 and Foxe3, all markers of lens induction; (3) by performing crosses between Tfr7 transgenic and Bmp7-null mice, it has been shown that there is a genetic interaction between Fgfr and Bmp7 signaling at the induction phases of lens development. This is manifested as exacerbated lens development defects and lower levels of Pax6 and Foxe3 expression in Tfr7/Tfr7, Bmp7^+/- mice, when compared with Tfr7/Tfr7 mice alone. Since Bmp7 is an established lens induction signal, this provides further evidence that Fgfr activity is important for lens induction. This analysis establishes a role for Fgfr signaling in lens induction and defines a genetic pathway in which Fgfr and Bmp7 signaling converge on Pax6 expression in the lens placode with the Foxe3 and Sox2 genes lying downstream (Faber, 2001).

The establishment of polarity is an important step during organ development. A function has been assigned for the paired and homeodomain transcription factor Pax6 in axis formation in the retina. Pax6 is a key factor of the highly conserved genetic network implicated in directing the initial phases of eye development. Pax6 is also essential for later aspects of eye development, such as lens formation and retinogenesis. Evidence that a highly conserved intronic enhancer, alpha, in intron 4 of the Pax6 gene is essential for the establishment of a distal^high-proximal^low gradient of Pax6 activity in the retina. In the mature retina, the activity mediated by the alpha-enhancer defines a population of retinal ganglion cells that project to two sickle-shaped domains in the superior colliculus and lateral geniculate nucleus. Deletion of the alpha-enhancer in vivo reveals that retinal Pax6 expression is regulated in two complementary topographic domains. Pax6 activity is required for the establishment, as well as the maintenance of dorsal and nasotemporal characteristics in the optic vesicle and, later, the optic cup (Bäumer, 2002).

Pax6 is a pivotal regulator of eye development throughout Metazoa, but the direct upstream regulators of vertebrate Pax6 expression are unknown. In vertebrates, Pax6 is required for formation of the lens placode, an ectodermal thickening that precedes lens development. The Meis1 and Meis2 homeoproteins are direct regulators of Pax6 expression in prospective lens ectoderm. In mice, Meis1 and Meis2 are developmentally expressed in a pattern remarkably similar to Pax6 and their expression is Pax6-independent. Biochemical and transgenic experiments reveal that Meis1 and Meis2 bind a specific sequence in the Pax6 lens placode enhancer that is required for its activity. Furthermore, Pax6 and Meis2 exhibit a strong genetic interaction in lens development, and Pax6 expression is elevated in lenses of Meis2-overexpressing transgenic mice. When expressed in embryonic lens ectoderm, dominant-negative forms of Meis down-regulate endogenous Pax6. These results contrast with those in Drosophila, where the single Meis homolog, Homothorax, has been shown to negatively regulate eye formation. Therefore, despite the striking evolutionary conservation of Pax6 function, Pax6 expression in the vertebrate lens is uniquely regulated (Zhang, 2002).

Despite the striking evolutionary conservation of the Pax6 pathway, it is interesting to contrast the current result with Drosophila, where the sole Meis homolog, Homothorax (Hth), suppresses eye formation in the ventral half of the eye and has been proposed to delimit the eye field. Meis/Homothorax function in controlling anterior-posterior embryonic patterning and proximo-distal limb development is conserved between Drosophila and vertebrates. Nevertheless, the fact that Meis1 and Meis2 are expressed throughout the developing lens, retina, and cornea further indicates that Meis1 and Meis2 cannot play exclusively repressive roles in vertebrate eye development. In addition, although the Meis binding site in the Pax6 lens enhancer is conserved from fish to human, it does not appear to be present in the eye imaginal disc enhancer of the Drosophila Pax6 homolog eyeless. In fact, when the eyeless enhancer was introduced into transgenic mice, it reproduced endogenous Pax6 expression in retina and spinal cord, but not in lens. Therefore, the mechanism of Pax6 regulation in the vertebrate lens placode differs in at least one respect from that in the Drosophila eye imaginal disc. Although Pax6 function is evolutionarily conserved, potentially indicating a monophyletic origin for the eye, it is attractive to suggest that divergent mechanisms regulating Pax6 expression also exist. These enhancer-specific differences may underlie the unique aspects of oculogenesis in vertebrates and invertebrates (Zhang, 2002).

The Pax6 gene plays crucial roles in eye development and encodes a transcription factor containing both a paired domain and a homeodomain. During embryogenesis, Pax6 is expressed in restricted tissues under the direction of distinct cis-regulatory regions. The head surface ectoderm-specific enhancer of mouse Pax6 directs reporter expression in the derivatives of the ectoderm in the eye, such as lens and cornea, but the molecular mechanism of its control remains largely unknown. A Pax6 protein-responsive element termed LE9 (52 bp in length) has been identified within the head surface ectoderm-specific enhancer. LE9, a sequence well conserved across vertebrates, acted as a highly effective enhancer in reporter analyses. Pax6 protein forms in vitro a complex with the distal half of LE9 in a manner dependent on the paired domain. The proximal half of the LE9 sequence contains three plausible sites of HMG domain recognition, and HMG domain-containing transcription factors Sox2 and Sox3 activate LE9 synergistically with Pax6. A scanning mutagenesis experiment indicates that the central site is most important among the three presumptive HMG domain recognition sites. Furthermore, Pax6 and Sox2 proteins form a complex when they are expressed together. Based on these findings, a model is proposed in which Pax6 protein directly and positively regulates its own gene expression, and Sox2 and Sox3 proteins interact with Pax6 protein, resulting in modification of the transcriptional activation by Pax6 protein (Aota, 2003).

Drosophila Pygopus was originally identified as a core component of the canonical Wnt signaling pathway and a transcriptional coactivator. This study investigated the microophthalmia that arises in mice with a germline null mutation of pygopus 2. This phenotype is a consequence of defective lens development at inductive stages. Using a series of regionally limited Cre recombinase transgenes for conditional deletion of Pygo2^flox, it has been shown that Pygo2 activity in pre-placodal presumptive lens ectoderm, placodal ectoderm and ocular mesenchyme all contribute to lens development. In each case, Pygo2 is required for normal expression levels of the crucial transcription factor Pax6. Finally, multiple lines of evidence are provided that although Pygo2 can function in the Wnt pathway, its activity in lens development is Wnt pathway-independent (Song, 2007).

A model is proposed for the function of Pygo2 in development of the lens. This is primarily a genetic model but can be superimposed on the tissue structures to indicate likely tissue interactions. In Pygo2^-/- embryos, reduced Pax6 immunofluorescence and Le-cre(GFP) reporter expression suggest that Pygo2 is upstream of the Pax6 ectoderm enhancer in regulating the placodal phase of Pax6 expression. By contrast, in the Pygo2 germline null, the pre-placodal phase of Pax6 expression (head surface ectoderm) is unchanged. In a reciprocal experiment, it was shown that in Pax6^Sey/Sey embryos (which represent the pre-placodal phase of Pax6), Pygo2 expression was unchanged. These data argue for a genetic model in which Pygo2 and Pax6^pre-placode converge on the EE to regulate Pax6 expression. Previous analysis has shown that Pax6^placode depends on the ectoderm enhancer and at least one additional enhancer. With the information currently available, the involvement of another Pax6 lens enhancer in Pygo2-dependent regulation cannot be excluded. The best candidate for a second lens enhancer in Pax6 is the SIMO element. It has been suggested that Pax6 can directly bind the ectoderm element. Pygo2 influence on the ectoderm element could be direct or indirect (Song, 2007).

Regional deletion of the Pygo2 conditional allele has indicated that Pygo2 in multiple tissues contributes to lens development. Deletion of Pygo2^flox with Wnt1-cre indicates that Pygo2 in neural crest-derived ocular mesenchyme positively influences lens development. In one model, this could occur by direct signaling of ocular mesenchyme to presumptive lens or, conceivably, indirectly through enhancement of the ability of the optic vesicle to induce lens. Either way, the end result is Pygo2-dependent upregulation of Pax6^placode. The more-severe lens phenotype occurring when Wnt1-cre is combined with the post-induction placodal ectoderm-driver Le-cre indicates that mesenchymal and placodal Pygo2 cooperate. A comparison of that outcome with the Ap2alpha-cre conditional (where deletion also occurs in pre-placodal ectoderm) suggests that this domain is also involved. This is consistent with a function for Pygo2 in parallel with Pax6^pre-placode. It is interesting to note that Pygo2 influences lens development through the ectoderm enhamcer, as do both Fgf receptor and Bmp7 signaling. It will be interesting to determine whether the non-Wnt activity of Pygo2 resides in one of these pathways (Song, 2007).

How transcription factors interpret the cis-regulatory logic encoded within enhancers to mediate quantitative changes in spatiotemporally restricted expression patterns during animal development is not well understood. Pax6 is a dosage-sensitive gene essential for eye development. This study identified the Prep1 (pKnox1) transcription factor, a homeobox gene of the TALE superfamily that was identified along with closely related Meis TFs as a regulator of the Pax6 pancreatic enhancer, as a critical dose-dependent upstream regulator of Pax6 expression during lens formation. Prep1 activates the Pax6 lens enhancer by binding to two phylogenetically conserved lower-affinity DNA-binding sites. Finally, a mechanism is described whereby Pax6 levels are determined by transcriptional synergy of Prep1 bound to the two sites, while timing of enhancer activation is determined by binding site affinity (Rowan, 2010).

Pax-6 transcriptional targets

The cell adhesion molecule L1 regulates axonal guidance and fasciculation during development. The regulatory region of the L1 gene has been identified and shown to be sufficient for establishing the neural pattern of L1 expression in transgenic mice. A DNA element identified within this region, called the HPD, contains binding motifs for both homeodomain and Pax proteins and responds to signals from bone morphogenetic proteins (BMPs). An ATTA sequence within the core of the HPD was required for binding to the homeodomain protein Barx2 while a separate paired domain recognition motif is necessary for binding to Pax-6. In cellular transfection experiments, L1-luciferase reporter constructs containing the HPD are activated an average of 4-fold by Pax-6 in N2A cells and 5-fold by BMP-2 and BMP-4 in Ng108 cells. Both of these responses are eliminated on deletion of the HPD from L1 constructs. In transgenic mice, deletion of the HPD from an L1-lacZ reporter results in a loss of beta-galactosidase expression in the telencephalon and mesencephalon. Collectively, these experiments indicate that the HPD regulates L1 expression in neural tissues via homeodomain and Pax proteins and is likely to be a target of BMP signaling during development (Meech, 1999).

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. The transcriptional promoter of the MMP gene, gelatinase B (gelB), has been characterized in transgenic mice, demonstrating the requirement for DNA sequences between 2522 and 119 for appropriate activity. Factors required for gelB promoter activity in the developing eye and reepithelializing adult cornea have been investigated. Adult mice (6-8 weeks of age) were anesthetized. Residual eye reflexes were blocked by topical application of an anaesthetic to the corneal surface. A circular demarcation was created in the central portion of one cornea of each mouse by excimer laser keratectomy to a depth of 40 mm, removing the epithelium, basement membrane, and anterior stroma to a diameter of 1.5 mm. This is similar to the photorefractive keratectomy procedure used in humans to correct refractive error. Antibiotic ointment was applied to the eyes after surgery. Mice were sacrificed 18 h after surgery at a time when the migrating epithelial sheet had almost resurfaced the corneal defect. Pax-6 antibodies were used in this study. Pax-6 is expressed in the adult eye. 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, two promoter regions were identified that mediate positive response to Pax-6. By electrophoretic mobility shift assay, two Pax-6 binding sites were further pinpointed within these response regions and direct interaction of the Pax-6 paired domain with one of these sites was demonstrated. These data suggest a mechanism by which Pax-6 may direct gelB expression in an eye-specific manner (Sivak, 2000).

Wnt signaling regulates a wide range of developmental processes such as proliferation, cell migration, axon guidance, and cell fate determination. The expression of secreted frizzled related protein-2 (SFRP-2), which codes for a putative Wnt inhibitor, in the developing nervous system, has been studied. SFRP-2 is expressed in several discrete neuroepithelial domains, including the diencephalon, the insertion of the eminentia thalami into the caudal telencephalon, and the pallial-subpallial boundary (PSB). Wnt-7b expression is similar to SFRP-2 expression. Because many of these structures are disrupted in Pax-6 mutant mice, SFRP-2 and Wnt-7b expression was examined in the forebrains of Pax-6 Sey/Sey mice. Pax-6 mutants were found to lack SFRP-2 expression in the PSB and diencephalon. Interestingly, Pax-6 mutants also lack Wnt-7b expression in the PSB, but Wnt-7b expression in the diencephalon is preserved. Furthermore, in the spinal cord of Pax-6 mutants, SFRP-2 and Wnt-7b expression is greatly reduced. These results suggest that by virtue of its apposition to Wnt-7b expression, SFRP-2 may modulate Wnt-7b function, particularly at boundaries such as the PSB, and that changes in Wnt signaling contribute to the phenotype of Pax-6 mutants (Kim, 2001).

This study examined how genetic pathways that specify neuronal identity and regulate neurogenesis interface in the vertebrate neural tube. Expression of the proneural gene Neurogenin2 (Ngn2) in the ventral spinal cord results from the modular activity of three enhancers active in distinct progenitor domains, suggesting that Ngn2 expression is controlled by dorsoventral patterning signals. Consistent with this hypothesis, Ngn2 enhancer activity is dependent on the function of Pax6, a homeodomain factor involved in specifying the identity of ventral spinal cord progenitors. Moreover, Ngn2 is required for the correct expression of Pax6 and several homeodomain proteins expressed in defined neuronal populations. Thus, neuronal differentiation involves crossregulatory interactions between a bHLH-driven program of neurogenesis and genetic pathways specifying progenitor and neuronal identity in the spinal cord (Scardigli, 2001).

Ngn2 is involved in crossregulatory interactions with homeodomain proteins involved in neuronal fate specification in the ventral spinal cord. In one direction, Ngn2 expression is driven by distinct enhancers that are active at different dorsoventral levels and that depend to various degrees on Pax6 function. In the other direction, Ngn2 activity is itself required for the proper expression of homeodomain proteins in progenitor domains and neuronal populations throughout the ventral spinal cord. The Ngn2 enhancers characterized in this study are active in progenitor domains that are restricted along the DV axis of the spinal cord, suggesting that Ngn2 expression may be regulated by Shh-dependent pathways that establish the DV positional identity of ventral progenitors. In support of this idea, the three Ngn2 enhancers examined are dependent to various degrees on the function of Pax6, a gene repressed by Shh in the ventral spinal cord, for the establishment of their distinct DV domains of activity. In the absence of Pax6, the activity of these enhancers is reduced or abolished in their normal domains, and is expanded to ectopic sites. Loss of Ngn2 expression and loss of E3 enhancer activity in the dorsal spinal cord of Sey embryos mutant for Pax6 is likely to reflect a function for Pax6 in this region of the neural tube, where it is normally expressed at low levels. Together, these data raise the intriguing possibility that Pax6 itself defines the DV position of Ngn2 enhancer activity (Scardigli, 2001).

The transcription factor Pax6 is required for eye morphogenesis in humans, mice and insects, and can induce ectopic eye formation in vertebrate and invertebrate organisms. Although the role of Pax6 has intensively been studied, only a limited number of genes have been identified that depend on Pax6 activity for their expression in the mammalian visual system. Using a large-scale in situ hybridization screen approach, a novel gene expressed in the mouse optic vesicle has been identified. This gene, Necab, encodes a putative cytoplasmic Ca²⁺-binding protein and coincides with Pax6 expression pattern in the neural ectoderm of the optic vesicle and in the forebrain pretectum. Remarkably, Necab expression is absent in both structures in Pax6 mutant embryos. By contrast, the optic vesicle-expressed homeobox genes Rx, Six3, Otx2 and Lhx2 do not exhibit an altered expression pattern. Using gain-of-function experiments, it has been shown that Pax6 can induce ectopic expression of Necab, suggesting that Necab is a direct or indirect transcriptional target of Pax6. In addition, Necab misexpression can induce ectopic expression of the homeobox gene Chx10, a transcription factor implicated in retina development. Taken together, these results provide evidence that Necab is genetically downstream of Pax6 and that it is a part of a signal transduction pathway in retina development (Bernier, 2001).

Expression of the proneural gene Neurogenin2 is controlled by several enhancer elements, with the E1 element active in restricted progenitor domains in the embryonic spinal cord and telencephalon that express the homeodomain protein Pax6. Pax6 function is both required and sufficient to activate this enhancer, and one evolutionary conserved sequence in the E1 element is identified with high similarity to a consensus Pax6 binding site. This conserved sequence binds Pax6 protein with low affinity both in vitro and in vivo, and its disruption results in a severe decrease in E1 activity in the spinal cord and in its abolition in the cerebral cortex. The regulation of Neurogenin2 by Pax6 is thus direct. Pax6 is expressed in concentration gradients in both spinal cord and telencephalon. The E1 element is activated only by high concentrations of Pax6 protein, and this requirement explains the restriction of E1 enhancer activity to domains of high Pax6 expression levels in the medioventral spinal cord and lateral cortex. By modifying the E1 enhancer sequence, it is also shown that the spatial pattern of enhancer activity is determined by the affinity of its binding site for Pax6. Together, these data demonstrate that direct transcriptional regulation accounts for the coordination between mechanisms of patterning and neurogenesis. They also provide evidence that Pax6 expression gradients are involved in establishing borders of gene expression domains in different regions of the nervous system (Scardigli, 2003).

A striking finding of this study is that the same mechanism is employed to control the expression of Ngn2 in progenitor domains located in two distant regions of the embryonic CNS, the ventral spinal cord and the dorsal telencephalon. Similarities in the molecular mechanisms that pattern the spinal cord and telencephalon along their dorsoventral axis have been noted before, and include common inductive signals such as Sonic Hedgehog and bone morphogenetic proteins, related intrinsic determinants, including HD proteins of the Pax and Nkx families, and bHLH proteins of the Mash and Ngn families, and in particular the establishment by Pax6 of boundaries between adjacent progenitor domains, through cross-regulatory interactions with the HD proteins Nkx2.2 in the spinal cord, and Nkx2.1 and Gsh2 in the telencephalon. The activity of the E1 enhancer in both spinal cord and telencephalon thus probably reflects a common role of Pax6 in these two territories. It must be noted however, that E1 is not active in all domains of high Pax6 expression [e.g., the retina), suggesting that regional determinants may act as co-factors to constrain Pax6 function and restrict E1 activity along the anteroposterior axis of the neural tube (Scardigli, 2003).

The generation of neurons by progenitors in the embryonic nervous system involves two distinct processes: the commitment of multipotent progenitors to a neuronal fate, resulting in their differentiation into neurons, and the specification of progenitor identity, resulting in the differentiation of neurons of a particular subtype. A number of studies suggest that these two processes are coupled at several levels. (1) Proneural bHLH genes, the major regulators of neuronal commitment in multipotent progenitors, are also involved in the specification of neuronal identity. In particular, proneural genes have been shown to control some aspects of the neuronal phenotype, such as the neurotransmission profile, through the regulation of downstream HD genes that directly activate genes encoding biosynthetic enzymes for neurotransmitters. (2) The regulation of the proneural genes themselves appears to be intimately linked with the regionalization of the neural tube, as these genes are expressed in restricted neuroepithelial domains with well-defined dorsoventral borders. Some of the genes that are involved in partitioning the neuroepithelium in dorsoventral progenitor domains have been shown to control the expression of proneural genes in these territories. For example, the HD protein Phox2b acts as a patterning gene to specify the identity of branchiomotor neuron progenitors in the hindbrain, and it simultaneously promotes the neuronal differentiation of these progenitors by upregulating the expression of the proneural genes Ngn2 and Mash1. A control of proneural gene expression by neural patterning genes has also been reported in Drosophila where the selector-like gene pannier regulates the notal pattern, and is the only factor to directly activate AS-C genes. Thus instances in which patterning genes control the expression of proneural genes are likely to be a general feature of neural development in both invertebrates and vertebrates (Scardigli, 2003).

This work provides the first demonstration that a proneural gene is directly regulated by a patterning gene in vertebrates, suggesting that neural patterning and neurogenesis may generally be tightly linked. It is likely that multiple patterning genes are involved in the generation of the complex expression patterns of proneural genes. Indeed, Pax6 is essential for the regulation of only one of the four known enhancer elements of Ngn2. Recent work suggests that in Drosophila, regulators of proneural genes act hierarchically rather than in a combinatorial manner, so that the number of direct transcriptional activators is actually very small. Further studies are necessary to determine whether this holds true for vertebrate proneural genes (Scardigli, 2003).

The telencephalon shows vast morphological variations among different vertebrate groups. The transcription factor neurogenin1 (ngn1) controls neurogenesis in the mouse pallium and is also expressed in the dorsal telencephalon of the evolutionary distant zebrafish. The upstream regions of the zebrafish and mammalian ngn1 loci harbour several stretches of conserved sequences. The upstream region of zebrafish ngn1 is capable of faithfully recapitulating endogenous expression in the zebrafish and mouse telencephalon. A single conserved regulatory region is essential for dorsal telencephalic expression in the zebrafish, and for expression in the dorsal pallium of the mouse. However, a second conserved region that is inactive in the fish telencephalon is necessary for expression in the lateral pallium of mouse embryos. This regulatory region, which drives expression in the zebrafish diencephalon and hindbrain, is dependent on Pax6 activity and binds recombinant Pax6 in vitro. Thus, the regulatory elements of ngn1 appear to be conserved among vertebrates, with certain differences being incorporated in the utilisation of these enhancers, for the acquisition of more advanced features in amniotes. These data provide evidence for the co-option of regulatory regions as a mechanism of evolutionary diversification of expression patterns, and suggest that an alteration in Pax6 expression was crucial in neocortex evolution (Blader, 2004).

To delineate the regulatory regions responsible for brain expression of ngn1 in older zebrafish embryos, transgenic lines carrying wild-type and deletion variants of ngn1 transgenes were analysed. Two regulatory regions were mapped that are required for transgene expression in the brain of post-somitogenesis-stage embryos. The first region, residing at position –6702 to –6490 bp upstream of the ATG start site, which harbours the LSE (lateral stripe element), drives expression in the dorsal telencephalon. A second regulatory region referred to as LATE was mapped to position –1775 to –1368. The LATE region, like the LSE, is highly conserved in mouse and human homologues of ngn1. Comparative functional studies were carried out in mouse embryos to investigate the activity of these conserved regulatory elements. Focus was placed on the dorsal telencephalon of the mouse, since this is undoubtedly the most derived brain region to have arisen during vertebrate evolution. The LSE drives expression in the dorsal telencephalon in both mouse and zebrafish embryos, indicating a conserved function with respect to telencephalic expression. Curiously, the LATE region of the zebrafish ngn1 gene drives expression in the lateral telencephalon of the mouse embryo but not in the zebrafish telencephalon. The area of activity of LATE overlaps with that of the paired-homeodomain transcription factor Pax6, suggesting a role of Pax6 in regulating the activity of LATE. Pax6 was shown to bind to a conserved Pax6-binding site in the LATE region. Moreover, the lack of pax6 activity in zebrafish by simultaneous knockdown of both pax6.1 and pax6.2 leads to a small eye phenotype and strongly reduces endogenous ngn1 and transgene expression. These results are consistent with a direct regulatory role of Pax6 on the activity of LATE. Based on the highly modular structure of vertebrate regulatory regions, which are usually composed of multiple short and degenerate binding sites for transcription factors, it is commonly assumed that elaboration of novel patterns of gene expression is accomplished by changes in the regulatory sequence. These data suggest that a pre-existing enhancer was co-opted, and that the evolution of the pax6 expression pattern led to the recruitment of LATE into the newly developed territories of the mouse telencephalon (Blader, 2004).

Pax-6 misexpression induces ectopic eyes

Misexpression of the transcription factor Pax6 in the vertebrate Xenopus laevis leads to the formation of differentiated ectopic eyes. A series of injection sites, RNA concentrations and times were tested and the phenotypic consequences examined in whole Xenopus embryos. It was found that when embryos were injected in one blastomere at the 16-cell stage or in two blastomeres at the 32-cell stage they displayed numerous eye-related phenotypes at a frequency of up to 50% when examined as tadpoles. The phenotypes were concentration-dependent and included (1) the formation of isolated ectopic lenses, (2) defects in the eye region proximal to the neural tube, and (3) the appearance of ectopic retinal pigment epithelium (RPE). In tadpoles, ectopic lenses are observed as pearl-like objects adjacent to the ectoderm. In section, these lenses show a lens epithelial layer and labele in the fiber cell region with antibodies to the lens-specific beta-crystallins. Multiple molecular markers indicate the presence of mature lens fiber cells, ganglion cells, Müller cells, photoreceptors and retinal pigment epithelial cells in a spatial arrangement similar to that of endogenous eyes. Lineage tracing experiments show that lens, retina and retinal pigment epithelium arise as a consequence of the cell-autonomous function of Pax6. These experiments also reveal that the cell autonomous activity of misexpressed Pax6 causes the ectopic expression of a number of genes including Rx, Otx2, Six3 and endogenous Pax6, each of which has been implicated in eye development. The formation of ectopic and endogenous eyes could be suppressed by coexpression of a dominant-negative form of Pax6. These data show that in vertebrates, as in the invertebrate Drosophila, Pax6 is both necessary and sufficient to trigger the cascade of events required for eye formation (Chow, 1999).

Ectopic eyes formed anywhere in the region anterior to the spinal cord-hindbrain junction and were more frequently located dorsally. In many, but not all cases of ectopic eyes, a retinal pigment epithelium-like layer was observed to extend from the central region of the ectopic eye to the brain in a similar manner to that observed for proximal eye defects seen with ectopic Pax6. Ectopic eyes induced by Pax6 misexpression display a similar morphology and organization to that of endogenous eyes. Histological sections through ectopic eyes of embryos at stage 48 reveal presumptive retinal ganglion cell, inner nuclear and outer nuclear layers separated by inner and outer plexiform layers. This suggests the presence of well differentiated neural retinas in ectopic eyes. As is the case in normal eyes, a retinal pigment epithelium- (PRE-) like layer is observed in close association with the outer (photoreceptor) cell layer of ectopic eyes. In some cases, the RPE-like layer surroundsthe entire eye cup while in others it surrounds only the retinal elements. Interestingly, a group of cells located at the distal tips of apparent neural retinas in ectopic eyes often displayed a tightly clustered arrangement reminiscent of that found in the ciliary margin zone of normal eyes. The ciliary margin zone is a region at the distal tip of normal, fully differentiated retinas that is made up of progenitor cells that can give rise to all retinal cell types. Combined, these data provide morphological evidence that Pax6 can induce ectopic eyes that contain all of the major cell layers found in normal eyes (Chow, 1999).

Embryos misexpressing Pax6 display numerous defects affecting the endogenous eye ranging from an extension of the RPE towards the midline to the juxtaposition of an expanded eye cup adjacent to the brain. Morphological and molecular data support the idea that these defects arose as a conversion of proximal eye fates to more distal fates characteristic of the eye cup. The demonstration that Pax6 functions cell autonomously in these phenotypes highlights the conclusion that Pax6 can direct ectopic eye formation in vertebrates and also sheds light on the role of Pax6 in defining the eye fields during normal eye development. Classical embryology has provided strong evidence for the existence of a single morphogenetic eye field that spans the midline of neural plate stage embryos. These and more recent studies have shown that signals derived from the underlying prechordal mesoderm and ventral forebrain resolve this single eye field into two. These studies have also shown that suppression of Pax6 expression in the midline occurs in the absence of cell migration and is under the control of the prechordal mesoderm signals. Interestingly, Pax6 is ectopically expressed in the proximal eye regions of cyclopic mutants in which this midline signaling is deficient. These observations, in combination with the presented gain-of-function data suggest that the regulation of Pax6 plays a critical role in defining the eye fields. Interestingly, eyes of mice having a null mutation in Pax2 bear a striking resemblance to the proximal eye defects seen in Xenopus embryos misexpressing Pax6. In these mice the RPE extends towards the midline and the ventral choroidal fissure fails to close. Both these phenotypes are observed in Xenopus embryos misexpressing Pax6 and suggest that Pax2 may function in part by inhibiting the Pax6-directed development of distal eye fates in the region of the optic stalk. Pax6 expressed from an injected RNA may bypass the normal function of Pax2 in specifying proximal eye fates in the optic stalk region. The proximal eye defects caused by Pax6 misexpression also resemble those in Xenopus embryos misexpressing Rx where extensions of RPE towards the midline are observed. Like Pax6, Rx is initially expressed in the anterior neural plate as a single band that resolves with time to the distal eye regions destined to become the optic vesicles. These data imply that Rx, like Pax6, can direct distal cell fate in the optic cup. The similarity in the phenotypic responses to Rx and Pax6 misexpression, combined with data showing that Pax6 can activate Rx expression argues that during development of optic cup-derived eye structures, Rx may function downstream of Pax6. This does not preclude the possibility that Rx may also have a function in regulating Pax6 in some cell types. Indeed, it has been shown recently that ectopic Pax6 expression is induced in embryos misexpressing Rx (Chow, 1999).

The data presented suggest that there is a cell-autonomous requirement for Pax6 in the development of both the lens and retinal components of ectopic eyes. This is consistent with results from analysis of chimeric mice, explant experiments and with the demonstration that Pax6-induced ectopic lens formation requires autonomous Pax6. Combined with previous data indicating that eye formation lies downstream of neural induction it is also reasonable to suggest that ectopic eyes may be formed through respecification of regions of the neural tube. In this vein, however, it is interesting to note that the Pax6 mutant mouse Small eye homozygote forms optic vesicle-like structures in the absence of functional Pax6 product. Although this indicates that the initiation of optic vesicle formation occurs in a Pax6-independent manner, the maintenance and continued development of these structures may be Pax6 dependent. Pax6 misexpression may recapitulate the genetic program responsible for optic vesicle formation. The lack of complete structure in ectopic eyes may therefore reflect the existence of eye-competent tissue that can respond to misexpressed Pax6 in the absence of normal optic vesicle morphology (Chow, 1999 and references).

Pax6 is essential for lens fiber cell differentiation

The developing ocular lens provides an excellent model system with which to study the intrinsic and extrinsic cues governing cell differentiation. Although the transcription factors Pax6 and Sox2 have been shown to be essential for lens induction, their later roles during lens fiber differentiation remain largely unknown. Using Cre/loxP mutagenesis, Pax6 and Sox2 were somatically inactivated in the developing mouse lens during differentiation of the secondary lens fibers and the regulatory interactions were explored of these two intrinsic factors with the canonical Wnt pathway. Analysis of the Pax6-deficient lenses revealed a requirement for Pax6 in cell cycle exit and differentiation into lens fiber cells. In addition, Pax6 disruption led to apoptosis of lens epithelial cells. Pax6 regulates the Wnt antagonist Sfrp2 in the lens, and Sox2 expression is upregulated in the Pax6-deficient lenses. However, this study demonstrates that the failure of differentiation following loss of Pax6 is independent of β-catenin signaling or Sox2 activity. This study reveals that Pax6 is pivotal for initiation of the lens fiber differentiation program in the mammalian eye (Shaham, 2009).

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