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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 Hensens 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).
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).
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