1: Genetics. 2004 May;167(1):233-41. The basic helix-loop-helix leucine zipper transcription factor Mitf is conserved in Drosophila and functions in eye development. Hallsson JH, Haflidadottir BS, Stivers C, Odenwald W, Arnheiter H, Pignoni F, Steingrimsson E. Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland. The MITF protein is a member of the MYC family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors and is most closely related to the TFE3, TFEC, and TFEB proteins. In the mouse, MITF is required for the development of several different cell types, including the retinal pigment epithelial (RPE) cells of the eye. In Mitf mutant mice, the presumptive RPE cells hyperproliferate, abnormally express the retinal transcriptional regulator Pax6, and form an ectopic neural retina. Here we report the structure of the Mitf gene in Drosophila and demonstrate expression during embryonic development and in the eye-antennal imaginal disc. In vitro, transcriptional regulation by Drosophila Mitf, like its mouse counterpart, is modified by the Eyeless (Drosophila Pax6) transcription factor. In vivo, targeted expression of wild-type or dominant-negative Drosophila Mitf results in developmental abnormalities reminiscent of Mitf function in mouse eye development. Our results suggest that the Mitf gene is the original member of the Mitf-Tfe subfamily of bHLH-Zip proteins and that its developmental function is at least partially conserved between vertebrates and invertebrates. These findings further support the common origin of the vertebrate and invertebrate eyes. PMID: 15166150 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Curr Biol. 2003 Jan 8;13(1):73-7. Ccd1, a novel protein with a DIX domain, is a positive regulator in the Wnt signaling during zebrafish neural patterning. Shiomi K, Uchida H, Keino-Masu K, Masu M. Department of Molecular Neurobiology, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, 305-8575, Tsukuba, Ibaraki, Japan. Wnt signaling plays a crucial role in directing cell differentiation, polarity, and growth. In the canonical pathway, Wnt receptors activate Dishevelled (Dvl), which then blocks the degradation of a key signal transducer, beta-catenin, leading to the nuclear accumulation of beta-catenin and induction of Wnt target genes through TCF/LEF family transcription factors. Here we identified a novel zebrafish gene encoding Ccd1, which possesses a DIX (Dishevelled-Axin) domain. DIX domains are essential for the signal transduction of two major Wnt downstream mediators, Dvl and Axin. Ccd1 formed homomeric and heteromeric complexes with Dvl and Axin and activated TCF-dependent transcription in vitro. In addition, overexpression of ccd1 in zebrafish embryos led to a reduction in the size of the eyes and forebrain (posteriorization), as seen with wnt8 overexpression, whereas a dominant-negative ccd1 (DN-ccd1) caused the opposite phenotype. Furthermore, the Wnt activation phenotype induced by ccd1 was inhibited by the expression of axin1 or DN-ccd1, and the wnt8 overexpression phenotype was rescued by DN-ccd1, suggesting that Ccd1 functions downstream of the Wnt receptor and upstream of Axin. These results indicate that Ccd1 is a novel positive regulator in this Wnt signaling pathway during zebrafish neural patterning. PMID: 12526749 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: J Struct Biol. 2000 Jul;131(1):56-66. Long-depth imaging of specific gene expressions in whole-mount mouse embryos with single-photon excitation confocal fluorescence microscopy and FISH. Palmes-Saloma C, Saloma C. National Institute of Molecular Biology and Biotechnology, University of the Philippines, Diliman, 1101, Philippines. Long-depth imaging of specific gene expression in the midgestation whole-mount mouse embryo (WME) is demonstrated with single-photon excitation (1PE) confocal fluorescence microscopy and fluorescence in situ hybridization. Expression domains of Pax-6 mRNA transcripts were labeled with an in situ hybridization probe that is a RNA sequence complementary to the cloned gene fragment and were rendered visible using two fluorochrome-conjugated antibodies that fluoresce at peak wavelengths of lambda(F) = 0.525 microm and lambda(F) = 0. 580 microm, respectively. Distributions of Pax-6 mRNA domains as deep as 1000 microm in the day 9.5 WME were imaged with a long-working-distance (13.6 mm) objective lens (magnification 5x). The scattering problem posed by the optically thick WME sample is alleviated by careful control of the detector pinhole size and the application of simple but fast postdetection image enhancement techniques, such as space and wavelength averaging to produce high-quality fluorescence images. A three-dimensional reconstruction that clearly shows the Pax-6 mRNA expression domains in the forebrain, diencephalon, optic cup, and spinal cord of the day 9.5 WME is obtained. The advantages of 1PE confocal fluorescence imaging over two-photon excitation fluorescence imaging are discussed for the case of long-depth imaging in highly scattering media. Imaging in midgestation WMEs at optical depths of more than 350 microm has not yet been realized with two-photon fluorescence excitation. Copyright 2000 Academic Press. PMID: 10945970 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Oncogene. 1999 Jan 28;18(4):1041-51. Involvement of poly (ADP-ribose)-polymerase in the Pax-6 gene regulation in neuroretina. Plaza S, Aumercier M, Bailly M, Dozier C, Saule S. CNRS EP 560/Institut Pasteur, Institut de Biologie, Lille, France. 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. PMID: 10023680 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Mol Cell Biol. 1995 Jun;15(6):3344-53. Quail Pax-6 (Pax-QNR) mRNAs are expressed from two promoters used differentially during retina development and neuronal differentiation. Plaza S, Dozier C, Turque N, Saule S. Laboratoire de Differenciation Cellulaire et Moleculaire, CNRS EP56, Institute Pasteur, Lille, France. During investigations on the regulation of the Pax-6 gene, we characterized a cDNA from quail neuroretina showing a 5' untranslated region distinct from that previously described and initiated from an internal promoter. Using RNase protection and primer extension mapping, we localized this second quail Pax-6 promoter, termed P1. As reported for the already described P0 promoter, P1 was also transactivated in vitro by the p46Pax-QNR protein. RNase protection assays performed with quail neuroretina RNA showed that P1-initiated mRNAs were detected before the P0-initiated mRNAs, remained constant up to embryonic day 8, and decreased slowly thereafter whereas, P0-initiated mRNAs accumulated up to embryonic day 8. In contrast, quail retinal pigmented epithelium expressed only the P1-initiated mRNAs. Transformation of these cells by the v-myc oncogene induced neuronal traits in the culture, which thereafter, in addition to the P1-initiated mRNAs, expressed 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. PMID: 7760830 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Mol Endocrinol. 1994 Jul;8(7):929-38. Pax-QNR/Pax-6, a paired box- and homeobox-containing gene expressed in neurons, is also expressed in pancreatic endocrine cells. Turque N, Plaza S, Radvanyi F, Carriere C, Saule S. Centre National de la Recherche Scientifique CNRS EP 56 Institut Pasteur de Lille, France. After differential screening of a cDNA library constructed from quail neuroretina cells infected with the v-myc containing avian retrovirus MC29, we have isolated a cDNA clone Pax-QNR, homologous to the murine Pax6 which is mutated in the autosomal dominant mutation small eye (Sey) of the mouse and aniridia in man. Here we report the characterization of Pax-QNR/Pax-6 expression in the chicken, quail, and mouse pancreas. In situ hybridization performed with E3 chick embryos demonstrated 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 we obtained rabbit antisera (paired domain, serum 11; domain between paired and homeo, serum 12; homeodomain, serum 13; and carboxyl-terminal part, serum 14) 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. We conclude that Pax-QNR/Pax-6 represents another gene expressed both in the endocrine pancreas and neuro-ectodermic tissues. PMID: 7984154 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------