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Regulation of avian Pax-6

A promoter region of the quail Pax-6 (Pax-QNR) gene has been characterized. Sequence analysis of the 5' flanking region reveals a TATA-like box and a CAAT box as well as several putative cis-regulatory elements. A 1.5-kilobase pair fragment, containing 1386 base pairs of 5' flanking sequence, the first exon, and a portion of the first intron, is able to efficiently promote expression of the bacterial CAT gene in quail neuroretina cells. Cotransfection of the Pax-QNR promoter with a vector expressing the 46 kilodalton Pax-QNR protein results in an increase in Pax-QNR promoter activity. The Pax-QNR protein is able to interact directly with the Pax-QNR promoter. Footprinting experiments have identified the binding sites for the Pax-QNR protein within the promoter region. These results show that Pax-QNR encodes a transcriptional activator and that it potentially trans-activates its own promoter (Plaza, 1993).

Differential screening of a cDNA library constructed from quail neuroretina cells infected with v-myc containing avian retrovirus MC29 isolated a cDNA clone, Pax-QNR, homologous to the murine Pax6. Pax-QNR/Pax-6 expression in the chicken, quail, and mouse pancreas has been characterized. In situ hybridization performed with E3 chick embryos demonstrates that, in addition to the documented expression of Pax-QNR/Pax-6 in the neural tube, this gene is also expressed in the pancreatic bud. This expression is later restricted to discrete parts of the organ. From bacterially expressed Pax-QNR peptides, rabbit antisera were obtained (paired domain, serum 11; domain between paired and homeo, serum 12; homeodomain, serum 13; and carboxyl-terminal part, serum 14) that are capable of specifically recognizing Pax-QNR/Pax-6 proteins (48, 46 kilodaltons) in cell lines derived from alpha- and beta-pancreatic cells, but not from exocrine derived cell lines. It is concluded that Pax-QNR/Pax-6 represents another gene expressed both in the endocrine pancreas and neuro-ectodermic tissues (Turque, 1994).

Using nuclear run-on assays, the tissue-specific expression of quail Pax-6 (Pax-QNR) P0-initiated mRNAs has been shown to be due in part to regulation of the gene at the transcriptional level. Regulatory sequences governing neuroretina-specific expression of the P0-initiated mRNAs were investigated. By using reporter-based expression assays, a region was characterized within the Pax-QNR gene, located 7.5 kbp downstream from the P0 promoter, that functions as an enhancer in neuroretina cells but not in nonexpressing P0-initiated mRNA cells (quail embryo cells and quail retinal pigment epithelial cells). This enhancer element functions in a position- and orientation-independent manner both on the Pax-QNR P0 promoter and the heterologous thymidine kinase promoter. Moreover, this enhancer element exhibits a developmental stage-specific activity during embryonic neuroretina development: in contrast to activity at day E7, the enhancer activity is very weak at day E5. This parallels the level of expression of P0-initiated mRNAs observed at the same stages. Using footprinting, gel retardation, and Southwestern (DNA-protein) analysis, the existence of four neuroretina-specific nuclear protein-binding sites, involving multiple unknown factors has been demonstrated. In addition, the quail enhancer element is structurally and functionally conserved in mice. All of these results strongly suggest that this enhancer element may contribute to the neuroretina-specific transcriptional regulation of the Pax-6 gene in vivo (Plaza, 1995a).

To understand the regulation of the Pax-6 gene, which plays an important role in eye development, the promoter region of the quail Pax-6 (Pax-QNR) gene has been characterized. In addition to TATA and CAAT boxes, sequence analysis reveals several putative cis-regulatory elements among which are three myb-responsive elements (MRE). C-myb encodes a nuclear, DNA-binding phosphoprotein that functions as transcriptional regulator. Co-transfection in quail embryo cells of the Pax-QNR/pax-6 promoter with a vector expressing the 75 kDa c-myb protein results in an increase in Pax-QNR promoter activity. Using footprinting experiments, multiple binding sites for the myb protein within the promoter region have been identified. Protein containing the myb DNA-binding domain fused to the VP16-transactivation domain is fully efficient in Pax-QNR promoter transactivation, demonstrating that myb can transactivate through a direct binding on DNA. However, a myb truncated protein devoid of DNA-binding domain is also able to transactivate the Pax-QNR promoter. These results show that this promoter can be transactivated by the myb protein directly as well as indirectly. c-myb is shown to be strongly expressed in the developing neuroretina, simultaneously with Pax-QNR. These observations suggest that the c-myb protein may be a regulator of Pax-QNR/pax-6 (Plaza, 1995b).

During investigations on the regulation of the Pax-6 gene, a cDNA from quail neuroretina was characterized showing a 5' untranslated region distinct from that previously described and initiated from an internal promoter. Using RNase protection and primer extension mapping, this second quail Pax-6 promoter, termed P1 was localized. Both the P0 and P1 promoter are transactivated in vitro by the p46 Pax-6 (Pax-QNR) protein. RNase protection assays performed with quail neuroretina RNA show that P1-initiated mRNAs are detected before the P0-initiated mRNAs, and remain constant up to embryonic day 8, decreasing slowly thereafter, whereas P0-initiated mRNAs accumulate up to embryonic day 8. In contrast, quail retinal pigmented epithelium expresses only the P1-initiated mRNAs. Transformation of these cells by the v-myc oncogene induces neuronal traits in the culture, which thereafter, in addition to the P1-initiated mRNAs, express Pax-QNR from the P0 promoter. These results suggest that expression of the quail Pax-6 gene is under the control of different regulators through alternate promoters, P0 being activated at the onset of neuronal differentiation (Plaza, 1995c).

The Pax-6 gene encodes a transcriptional master regulator involved in the development of the eye. The quail Pax-6 gene is expressed in the neuroretina from two promoters, P0 and P1, P0 being activated at the onset of neuronal differentiation. Two regions in the quail Pax-6 gene 5' flanking sequences, located 6 and 2.5 kbp upstream from the P0 promoter have been identified that, like the previously characterised intragenic enhancer (EP enhancer), function as neuroretina-specific enhancers whose activity is restricted to the P0 promoter. Moreover, the activity of these 5' enhancers in embryonic neuroretina cells is weaker at day 5 than at day 7, like the EP enhancer, and parallels the level of expression of P0-initiated mRNAs. Footprinting experiments show that neuroretina-specific factors bind to these 5' enhancer elements. In addition, these quail Pax-6 enhancer elements, as well as the P0 promoter, are structurally and functionally conserved in humans. These results strongly suggest that these enhancer elements may contribute to the neuroretina-specific transcriptional regulation of the Pax-6 gene in vivo. Thus the complex regulation of the quail Pax-6 gene is also conserved in humans (Plaza, 1999).

Pax-QNR/Pax-6 products are expressed in the avian neuroretina. Five Pax-6 proteins (48, 46, 43, 33, and 32 kDa) have been characterized, among which the 33 and 32 kDa proteins are devoid of the paired domain. In contrast to the 48-kDa (containing an alternative paired exon 4a) and 46-kDa proteins exclusively located in the nucleus, the 43- (in which the paired exon 5 is spliced out), 33-, and 32-kDa proteins were also found in the cytoplasmic compartment. Two nuclear targeting sequences are reported: the basic LKRKLQR region (amino acids 206-212) located in the NH2 terminus of the homeodomain used by the p43 and 33/32 kDa proteins; and the paired exon 5 sequence. Recently reported has been a case of human aniridia, where arginine 208 of LKRKLQR is mutated into a tryptophan. This mutation was introduced into the Pax-QNR p46, p43, and p33/32 proteins. No effect on the nuclear localization or in transactivation potential of the proteins could be observed. Among the several Pax-QNR isoforms characterized, only p46 exhibits DNA-binding and transactivating properties on the Pax-QNR promoter. Deletions of parts of the protein show that the Pax-6 transactivation domain is located in the carboxyl terminus of the protein (Carriere, 1995).

Pax6 is a paired-type homeobox gene expressed in discrete regions of the central nervous system. In the spinal cord of 7- to 10-somite-stage chicken embryos, Pax6 is not detected within the caudal neural plate, but is progressively upregulated in the neuroepithelium neighbouring each newly formed somite. This initial activation of Pax6 is controlled via the paraxial mesoderm in correlation with somitogenesis. High levels of Pax6 expression occur independent of the presence of SHH-expressing cells when neural plates are maintained in culture in the presence of paraxial mesoderm. Grafting a somite caudally under a neural plate that has not yet expressed the gene induces a premature activation of Pax6. Furthermore, after the graft of a somite, a period of incubation corresponding to the individualization of a new somite in the host embryo produces an appreciable activation of Pax6. Conversely, Pax6 expression is delayed under conditions where somitogenesis is retarded, i.e., when the rostral part of the presomitic mesoderm is replaced by the same tissue isolated more caudally. Finally, Pax6 transcripts disappear from the neural tube when a somite is replaced by presomitic mesoderm, suggesting that the somite is also involved in the maintenance of Pax6 expression in the developing spinal cord. All together these observations lead to the proposal that Pax6 activation is triggered by the paraxial mesoderm in phase with somitogenesis in the cervical spinal cord. With respect to the role of SHH in Pax6 induction, prospective neural plates isolated caudal to Hensen’s node and maintained in vitro display Pax6 expression in the absence of SHH producing notochordal and floor plate cells. Also, the presence of the notochord expressing SHH is not sufficient to upregulate and maintain Pax6 expression in the cervical spinal cord after removal of a somite. Consequently, it is proposed that mechanisms others than SHH signaling may be involved in regulating Pax6 expression in the neural tube. Molecular evidence suggests that a developmental clock may be linked to somitogenesis of the paraxial mesoderm. The developing spinal cord has no obvious anteroposterior landmarks, but genes such as Pax6 are activated at precise times and locations along the rostrocaudal axis and such an activation correlates with somitogenesis. It is therefore tempting to speculate that, at least in some regions of the developing spinal cord, somitogenesis may be used as a clock to activate specific genes in a temporally and spatially appropriate manner. Together, these data argue in favour of a model in which Pax6 is activated in the cervical spinal cord via a positive signal from the somite, this signal being maintained at least for the next few hours to stabilize the gene expression. The nature of the signaling molecule mediating Pax6 upregulation remains unknown. The fact that a preincubation of the somite in blocking anti-SHH antibodies does not abolish the activity of the somite suggests that the factor is not SHH, even if this molecule is well known for its ability to upregulate Pax6 expression (Pituello, 1999)

Transcriptional targeting by avian Pax-6

Pax-QNR, cloned from the quail, is homologous to the murine Pax-6. The 46 kDa Pax-QNR protein binds specifically to the e5 DNA recognition sequence present upstream of the Drosophila even-skipped gene. The Pax-QNR paired and homeobox domains expressed separately in bacteria are both able to recognize this sequence. The core sequence recognized by the paired domain of Pax genes is TTCC (GGAA), and this sequence is also present in the core recognition site bound specifically by Ets family-encoded proteins. Ets proteins are a family of transcription factors sharing a highly conserved 85 amino acid DNA binding domain. Pax-QNR/Pax-6 expressed in reticulocyte lysate is able to specifically recognize several Ets binding sites. In addition, the transactivation mediated by the Ets-1 through the Polyomavirus enhancer sequence is specifically inhibited by the Pax-QNR in transient transfection assay (Plaza, 1994).

In quail neuroretinas, it has been observed that Engrailed (En-1) is expressed both in the ganglionic and the amacrine cell layers, similar to the expression patterns of Pax-6. Because a decrease of Pax-6 expression is observed in the neuroretina of hatched animals, the effect of the chicken En-1 and En-2 proteins on Pax-6 expression was examined. En-1 and to some extent En-2 are able to repress the basal and the p46Pax-6-activated transcription from the two Pax-6 promoters. Infection of retinal pigmented epithelium by a virus encoding the En-1 protein represses the endogenous Pax-6, and a similar effect is observed with a homeodomain-deleted En-1. In vitro interaction indicates that En proteins are able to interact with the p46Pax-6 through the paired domain. This interaction negatively regulates the DNA-binding properties of the p46Pax-6. These results suggest an interplay between En-1 and Pax-6 during the central nervous system development and indicate that En-1 may be a negative regulator of Pax-6 (Plaza, 1997).

Regionalization of a simple neural tube is a fundamental event during the development of the central nervous system. To analyze in vivo the molecular mechanisms underlying the development of the mesencephalon, expressed Engrailed, which is expressed in developing mesencephalon, was ectopically expressed in the brain of chick embryos by in ovo electroporation. Misexpression of Engrailed causes a rostral shift of the di-mesencephalic boundary, and causes transformation of dorsal diencephalon into tectum, a derivative of dorsal mesencephalon. Ectopic Engrailed rapidly represses Pax-6, a marker for diencephalon, which precedes the induction of mesencephalon-related genes, such as Pax-2, Pax-5, Fgf8, Wnt-1 and EphrinA2. In contrast, a mutant Engrailed, En-2(F51 to E), bearing mutation in the EH1 domain, which has been shown to interact with a co-repressor, Groucho, does not show the phenotype induced by wild-type Engrailed. Furthermore, VP16- Engrailed chimeric protein, the dominant positive form of Engrailed, causes a caudal shift of di-mesencephalic boundary and ectopic Pax-6 expression in mesencephalon. These data suggest that (1) Engrailed defines the position of dorsal di-mesencephalic boundary by directly repressing diencephalic fate, and (2) Engrailed positively regulates the expression of mesencephalon-related genes by repressing the expression of their negative regulator(s) (Araki, 1999).

It has been demonstrated previously that Pax-6, a paired domain (PD)/homeodomain (HD) transcription factor critical for eye development, contributes to the activation of the alphaB-, alphaA-, delta1-, and zeta-crystallin genes in the lens. The possibility was examined that the inverse relationship between the expression of Pax-6 and beta-crystallin genes within the developing chicken lens reflects a negative regulatory role for Pax-6. Cotransfection into primary embryonic chicken lens epithelial cells or fibroblasts of a plasmid containing the betaB1-crystallin promoter fused to the chloramphenicol acetyltransferase reporter gene and a plasmid containing the full-length mouse Pax-6 coding sequences represses the activity of this promoter by as much as 90%. Pax-6 constructs lacking the C-terminal activation domain repress betaB1-crystallin promoter activity as effectively as the full-length protein, but the PD alone or Pax-6 (5a), a splice variant with an altered PD affecting its DNA binding specificity, do not. DNase footprinting analysis reveals that truncated Pax-6 (PD+HD) binds to three regions (-183 to -152, -120 to -48, and -30 to +1) of the betaB1-crystallin promoter. The betaB1-crystallin promoter sequence from -120 to -48 contains a cis element (PL2 at -90 to -76) that stimulates the activity of a heterologous promoter in lens cells but not in fibroblasts. Pax-6 binds to PL2 and represses its ability to activate promoter activity; moreover, mutation of PL2 eliminates binding by Pax-6. Taken together, these data indicate that Pax-6 (via its PD and HD) represses the betaB1-crystallin promoter by direct interaction with the PL2 element. It is suggested that the relatively high concentration of Pax-6 contributes to the absence of betaB1-crystallin gene expression in lens epithelial cells and that diminishing amounts of Pax-6 in lens fiber cells during development allow activation of this gene (Duncan, 1998).

Paracrine Pax6 activity regulates oligodendrocyte precursor cell migration in the chick embryonic neural tube

Homeoprotein transcription factors play fundamental roles in development, ranging from embryonic polarity to cell differentiation and migration. Research in recent years has underscored the physiological importance of homeoprotein intercellular transfer in eye field development, axon guidance and retino-tectal patterning, and visual cortex plasticity. This study used the embryonic chick neural tube to investigate a possible role for homeoprotein Pax6 transfer in oligodendrocyte precursor cell (OPC) migration. The extracellular expression of Pax6 are reported in this study along with the effects of gain and loss of extracellular Pax6 activity on OPCs. Open book cultures with recombinant Pax6 protein or Pax6 blocking antibodies, as well as in ovo gene transfer experiments involving expression of secreted Pax6 protein or secreted Pax6 antibodies, provide converging evidences that OPC migration is promoted by extracellular Pax6. The paracrine effect of Pax6 on OPC migration is thus a new example of direct non-cell autonomous homeoprotein activity (Di Lullo, 2011).

Table of contents

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

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