Biomedical subjects

S Saule

Publications and source records attributed to S Saule.

At least 19 recordsLinked to original sources

PubMed2005

Comparative analysis of gnathostome Otx gene expression patterns in the developing eye: implications for the functional evolution of the multigene family.

We have performed a detailed analysis of the expression pattern of the three gnathostome Otx classes in order to gain new insights into their functional evolution. Expression patterns were examined in the developing eye of a chondrichthyan, the dogfish, and an amniote, the chick, and compared with the capacity of paralogous proteins to induce a pigmented phenotype in cultured retina cells in cooperation with the bHLH-leucine zipper protein Mitf. This analysis indicates that each Otx class is characterized by highly specific and conserved expression features in the presumptive RPE, where Otx1 and Otx2, but not Otx5, are transcribed at optic vesicle stages, in the differentiating neural retina, where Otx2 and Otx5 show a conserved dynamic expression pattern, and in the forming ciliary process, a major site of Otx1 expression. Furthermore, the paralogous proteins of the dogfish and the mouse do not display any significant difference in their capacity to induce a pigmented phenotype, suggesting a functional equivalency in the specification and differentiation of the RPE. These data indicate that specific functions selectively involving each Otx orthology class were fixed prior to the gnathostome radiation and highlight the prominent role of regulatory changes in the functional diversification of the multigene family.

Animals↗

PubMed2005

[Normal and malignant choroidal melanocytes: from cell to clinical approach].

The molecular and cellular basis of human choroidal malignant melanoma progression has remained largely unknown. However, choroidal melanoma is the most important primary intraocular tumor in adults. Developmentally, choroidal melanocytes are of neural crest origin similar to cutaneous melanocytes. However, there are some significant differences between cutaneous and uveal melanocytes that have yet to be fully assessed. The purpose of this study is to describe choroidal melanocytes. We will describe the significant differences between cutaneous and uveal melanocytes as well as the congenital and acquired diseases of uveal melanocytes. We will then describe the cellular and molecular mechanisms involved in melanoma progression.

Cell Proliferation↗

PubMed2001

Interaction of Maf transcription factors with Pax-6 results in synergistic activation of the glucagon promoter.

In the endocrine pancreas, alpha-cell-specific expression of the glucagon gene is mediated by DNA-binding proteins that interact with the G1 proximal promoter element. Among these proteins, the paired domain transcription factor Pax-6 has been shown to bind to G1 and to transactivate glucagon gene expression. Close to the Pax-6-binding site, we observed the presence of a binding site for a basic leucine zipper transcription factor of the Maf family. In the present study, we demonstrate the presence of Maf family members in the endocrine pancreas that bind to G1 and transactivate glucagon promoter expression. In transient transfection experiments, we found that the transactivating effect on the glucagon promoter was greatly enhanced by the simultaneous expression of Maf transcription factors and Pax-6. This enhancement on glucagon transactivation could be correlated with the ability of these proteins to interact together but does not require binding of Maf proteins to the G1 element. Furthermore, we found that Maf enhanced the Pax-6 DNA binding capacity. Our data indicate that Maf transcription factors may contribute to glucagon gene expression in the pancreas.

Animals↗

PubMed2001

Specific Pax-6/microphthalmia transcription factor interactions involve their DNA-binding domains and inhibit transcriptional properties of both proteins.

Pax-6 and microphthalmia transcription factor (Mitf) are required for proper eye development. Pax-6, expressed in both the neuroretina and pigmented retina, has two DNA-binding domains: the paired domain and the homeodomain. Mice homozygous for Pax-6 mutations are anophthalmic. Mitf, a basic helix-loop-helix leucine zipper (b-HLH-LZ) transcription factor associated with the onset and maintenance of pigmentation, identifies the retinal pigmented epithelium during eye development. Loss of Mitf function results in the formation of an ectopic neuroretina at the expense of the dorsal retinal pigmented epithelium. In the present study, we investigated the interaction between Pax-6 and Mitf. In transient transfection-expression experiments, we found that transactivating effects of Pax-6 and Mitf on their respective target promoters were strongly inhibited by co-transfection of both transcription factors. This repression was due to direct protein/protein interactions involving both Pax-6 DNA-binding domains and the Mitf b-HLH-LZ domain. These results suggest that Pax-6/Mitf interactions may be critical for retinal pigmented epithelium development.

Animals↗

PubMed2001

The AP-3-dependent targeting of the melanosomal glycoprotein QNR-71 requires a di-leucine-based sorting signal.

The Quail Neuroretina clone 71 gene (QNR-71) is expressed during the differentiation of retinal pigmented epithelia and the epidermis. It encodes a type I transmembrane glycoprotein that shares significant sequence homologies with several melanosomal proteins. We have studied its intracellular traffic in both pigmented and non-pigmented cells. We report that a di-leucine-based sorting signal (ExxPLL) present in the cytoplasmic domain of QNR-71 is necessary and sufficient for its proper targeting to the endosomal/premelanosomal compartments of both pigmented and non-pigmented cells. The intracellular transport of QNR-71 to these compartments is mediated by the AP-3 assembly proteins. As previously observed for the lysosomal glycoproteins Lampl and LimpII, overexpression of QNR-71 increases the amount of AP-3 associated with membranes, and inhibition of AP-3 synthesis increases the routing of QNR-71 towards the cell surface. In addition, expression of QNR-71 induces a misrouting of endogenous LampI to the cell surface. Thus, the targeting of QNR-71 might be similar to that of the lysosomal integral membrane glycoproteins LampI and LimpII. This suggests that sorting to melanosomes and lysosomes requires similar sorting signals and transport machineries.

Adaptor Proteins, Vesicular Transport↗

PubMed2001

[The myc oncogene and transdifferentiation of the retinal pigment epithelium].

The retinal pigment epithelium (RPE) develops from the same sheet of neuroepithelium as the neuroretina. When infected with MC29, a v-myc expressing virus, the RPE cells can be induced to transdifferentiate and to take a neuroretinal epithelium fate. After a PCR-based differential screening from these cells we have identified three genes of interest. Qath5, a quail basic helix-loop-helix (bHLH) gene that is closely related to the Drosophila atonal, and whose expression is found in the developing neuroretina. A Chx10-related homeobox gene also expressed in the developing neuroretina and HuD, a RNA-binding protein not expressed in the RPE but expressed during neurogenesis. Beside these genes whose function is involved in regulating neuronal differentiation myc also induced a transient Mitf expression. Mitf is expressed in the entire optic cup, later restricted to the pigmented retina. Mitf is involved in the regulation of the pigmented differentiation. We conclude that v-myc can reverse the RPE to the bipotential retinal primordia.

Alpharetrovirus↗

PubMed1999

Evidence that POU factor Brn-3B regulates expression of Pax-6 in neuroretina cells.

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, and is regulated by an intragenic neuroretina-specific enhancer (EP enhancer). The activity of this enhancer is restricted to the P0 promoter, which is activated at the onset of neuronal differentiation. In this article, we show that the POU domain transcription factor Brn-3b, which is expressed in various regions of the brain including retina and sensory neurons, is one of the factors interacting with the EP enhancer. Brn-3b strongly activates the EP enhancer in neuroretina cells but not in other cell types. Interestingly, this activation appears to be specific for Brn-3b, as the closely related POU factors Brn-3a and Brn-3c do not show activation of the EP enhancer. Our results identify the Pax-6 gene as a new potential downstream effector of the POU transcription factor Brn-3b.

Animals↗

PubMed1999

Pax-6 and Cdx-2/3 interact to activate glucagon gene expression on the G1 control element.

The promoter element G1, critical for alpha-cell-specific expression of the glucagon gene, contains two AT-rich sequences important for transcriptional activity. Pax-6, a paired homeodomain protein previously shown to be required for normal alpha-cell development and to interact with the enhancer element G3 of the glucagon gene, binds as a monomer to the distal AT-rich site of G1. However, although the paired domain of Pax-6 is sufficient for interaction with the G3 element, the paired domain and the homeodomain are required for high affinity binding to G1. In addition to monomer formation, Pax-6 interacts with Cdx-2/3, a caudal-related homeodomain protein binding to the proximal AT-rich site, to form a heterodimer on G1. Both proteins are capable of directly interacting in the absence of DNA. In BHK-21 cells, Pax-6 activates glucagon gene transcription both through G3 and G1, and heterodimerization with Cdx-2/3 on G1 leads to more than additive transcriptional activation. In glucagon-producing cells, both G1 and G3 are critical for basal transcription, and the Pax-6 and Cdx-2/3 binding sites are required for activation. We conclude that Pax-6 is not only critical for alpha-cell development but also for glucagon gene transcription by its independent interaction with the two DNA control elements, G1 and G3.

Animals↗

PubMed1999

Involvement of poly (ADP-ribose)-polymerase in the Pax-6 gene regulation in neuroretina.

The quail Pax-6 gene is expressed from two promoters named P0 and P1. P0 promoter is under the control of a neuroretina-specific enhancer (EP). This enhancer activates the P0 promoter specifically in neuroretina cells and in a developmental stage-dependent manner. The EP enhancer binds efficiently, as revealed by southwestern experiments, to a 110 kDa protein present in neuroretina cells but not in Quail Embryos Cells and Retinal Pigmented Epithelium which do not express the P0-initiated mRNAs. To study the role of p110 in Pax-6 regulation, we have purified the p110 from neuroretina cells extracts. Based on the peptide sequence of the purified protein, we have identified the p110 as the poly(ADP-ribose) polymerase (PARP). Using bandshift experiments and footprinting studies, we present evidence that PARP is a component of protein complexes bound to the EP enhancer that increases the on rate of the protein complex formation to DNA. Using PARP inhibitors (3AB and 6.5 Hphe), we show that these products are able to inhibit EP enhancer activity in neuroretina cells. Finally, we demonstrate that these inhibitors are able to decrease the expression of the P0-initiated mRNA in the MC29-infected RPE cells which, in contrast to the RPE cells, accumulated the PARP in response to v-myc expression. Our results suggest that PARP is involved in the Pax-6 regulation.

Animals↗

PubMed1999

High conservation of cis-regulatory elements between quail and human for the Pax-6 gene.

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. In this paper we have identified two regions in the quail Pax-6 gene 5' flanking sequences, located 6 and 2.5 kbp upstream from the P0 promoter 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 we show that 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.

Animals↗

PubMed1999

Expression of the microphthalmia-associated basic helix-loop-helix leucine zipper transcription factor Mi in avian neuroretina cells induces a pigmented phenotype.

The microphthalmia gene (mi) appears to be required for pigment cell development, based on its mutation in mi mice. The mi gene encodes a basic helix-loop-helix leucine zipper transcription factor (Mi) with tissue-restricted expression. To investigate the role of mi in cell proliferation and pigmentation, we transfected neuroretina (NR) cells with a recombinant virus expressing the murine mi cDNA. The virus induced the proliferation of chicken NR cells in response to fibroblast growth factor 2, which enabled them to form colonies in soft agar. In contrast to control cultures, transfected chicken NR cells or quail NR cells became rapidly pigmented and strongly expressed the QNR-71 mRNA encoding a melanosomal protein. These results demonstrate that Mi not only acts as pigmentation inducer but is also able to modulate the response of cells to growth factors.

Amino Acid Sequence↗

PubMed1997

Overexpression of A-myb induces basic fibroblast growth factor-dependent proliferation of chicken neuroretina cells.

A-Myb behaves similarly to c-Myb in chicken neuroretina cells in its ability to induce fibroblast-like differentiation, to promote growth in the presence of basic fibroblast growth factor (bFGF), and to induce Pax-6 and mim-1 expression. The one difference between c-Myb and A-Myb in these cells is that the former but not the latter protein causes colony formation in soft agar in the presence of bFGF.

Acetyltransferases↗

PubMed1997

The homeobox-containing Engrailed (En-1) product down-regulates the expression of Pax-6 through a DNA binding-independent mechanism.

By in situ hybridization of quail neuroretinas, we observed that Engrailed (En-1) is expressed both in the ganglionic and the amacrine cell layers, similarly to Pax-6. Because we observed a decrease of Pax-6 expression in the neuroretina of hatched animals, we studied the effect of the chicken En-1 and En-2 proteins on Pax-6 expression. En-1 and to some extent En-2 were 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 repressed the endogenous Pax-6, and a similar effect was 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.

Animals↗

PubMed1996

Characterization of a new melanocyte-specific gene (QNR-71) expressed in v-myc-transformed quail neuroretina.

Quail neuroretina cells (QNR) infected with the v-myc-expressing retrovirus MC29 become pigmented after several passages in vitro. After differential screening of a cDNA library constructed from these cells, we have isolated a cDNA clone (QNR-71) which identifies an RNA expressed only in the pigmented layer of the retina and in the epidermis. This gene can also be induced in other cell types transformed by MC29, suggesting that QNR-71 may be regulated by the v-myc protein. Sequence analysis showed that the QNR-71 cDNA exhibits stretches of homologies with melanosomal proteins encoding genes. From bacterially expressed QNR-71 peptides we obtained rabbit antisera able to specifically recognize two proteins of 95 and 100 kDa in pigmented retinal cells, but not in the neuroretina. To study the regulation of QNR-71, we used promoter fragments linked to the CAT reporter gene, in transient co-expression assay. We observed an increase in CAT expression with a c-MYC and microphtalmia (mi) expression vectors. Both MYC and mi activate the QNR-71 promoter through direct binding to a CATGTG site present in the promoter fragment.

Amino Acid Sequence↗

PubMed1996

Back-mutation of the V-Ets to the C-Ets carboxy-terminal amino acids in the P135gag-myb-ets results in chicken neuroretina cells transformation and loss of basic fibroblast growth factor responsiveness.

The v-Myb, v-Ets containing E26 retrovirus (called in this work E26ABC) induces the proliferation of chicken neuroretina (CNR) cells in minimal medium, strongly stimulated by basic Fibroblast Growth Factor (bFGF) which confers on them the ability to form colonies in soft agar. V-Ets differs from its cellular counterpart c-Ets-1 by two point mutations and by the replacement of the 13 last C-terminal amino acids by 16 unrelated residues as a consequence of DNA segment inversion in the viral sequence. It has been documented that this different C-terminal sequence influences DNA binding activity and specificity. Replacement in E26ABC virus of the sequence encoding the 16 v-Ets last C-terminal amino acids by the sequence encoding the 13 c-Ets-1 derived C-terminus (virus E26ABO), results in the production of a P135gag-myb-ets with modified biological properties on CNR cells. E26ABO infected CNR cells proliferate in minimal medium more efficiently than E26ABC, are unresponsive to bFGF and able to grow in soft agar. In contrast, CNR cells infected by viruses encoding Myb and Ets proteins either in the E26ABO or in the E26ABC configuration are bFGF responsive. Since Myb alone is sufficient to induce bFGF responsiveness on CNR cells, these results suggest that the c-Ets-1 C-terminus interferes with the Myb activity of the E26ABO P135gag-myb-ets protein in CNR cells.

Animals↗

PubMed1996

An identical effect mediated by thyroid deficiency or oncogene v-erbA in the chick embryo.

We have shown earlier that the association of v-myc and v-erbA (MAHEVA construct) is responsible for the appearance of a specific phenotype in chick embryos inoculated at E3. This phenotype comprises rapidly growing heart rhabdomyomas (induced by v-myc alone) and within these tumors secondarily appearing cartilage nodules (Bachnou et al., Oncogene 6: 1041-1047, 1991). Here we report that v-erbA can be replaced by thyroid deficiency. When decapitated embryos were inoculated with virus MC29 (v-myc alone) or when v-myc inoculated embryos were treated with thiourea, 100% of the embryos reaching E17 to E19 displayed tumoral hearts bearing cartilage nodules. We thus report in vivo evidence that v-erbA acts by antagonizing the effects of thyroid hormones. Remarkably, thyroid deficiency rendered embryos more sensitive to the effect of v-myc, since 100% developed heart rhabdomyomas and cartilage nodules, versus about 70% affected when either v-myc or MAHEVA were inoculated. Thyroid deficiency did not alter the species-specific character of transdifferentiation, since only chick but not quail embryos developed cartilage nodules after thyroidectomy or MAHEVA infection.

Animals↗

PubMed1995

C-Myb acts as transcriptional activator of the quail PAX6 (PAX-QNR) promoter through two different mechanisms.

To understand the regulation of the Pax-6 gene, which plays an important role in eye development, we have characterized the promoter region of the quail Pax-6(Pax-QNR) gene. In addition to TATA and CAAT boxes, sequence analysis revealed several putative cis-regulatory elements among which 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 resulted in an increase in Pax-QNR promoter activity. By footprinting experiments we identified multiple binding sites for the myb protein within the promoter region. Protein containing the myb DNA-binding domain fused to the VP16-transactivation domain was 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 was 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. Finally we show by in situ hybridization that c-myb is 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.

Animals↗

PubMed1995

Identification and characterization of a neuroretina-specific enhancer element in the quail Pax-6 (Pax-QNR) gene.

Using nuclear run-on assays, we showed that the tissue-specific expression of quail Pax-6 (Pax-QNR) P0-initiated mRNAs is 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, we characterized a region 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 functioned in a position- and orientation-independent manner both on the Pax-QNR P0 promoter and the heterologous thymidine kinase promoter. Moreover, this enhancer element exhibited a developmental stage-specific activity during embryonic neuroretina development: in contrast to activity at day E7, the enhancer activity was very weak at day E5. This paralleled the level of expression of P0-initiated mRNAs observed at the same stages. Using footprinting, gel retardation, and Southwestern (DNA-protein) analysis, we demonstrated the existence of four neuroretina-specific nuclear protein-binding sites, involving multiple unknown factors. In addition we showed that 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.

Animals↗