1: Dev Neurosci. 2005;27(6):364-77. Gliogenic radial glial cells show heterogeneity in the developing mouse spinal cord. Ogawa Y, Takebayashi H, Takahashi M, Osumi N, Iwasaki Y, Ikenaka K. Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, Okazaki, Japan. The central nervous system of the mammalian embryo is organized according to the expression of region-specific transcription factors along the anteroposterior and/or the dorsoventral axis. For example, the dorsal ventricular zone (VZ) of the embryonic spinal cord expresses Pax3 and Pax7, the ventral VZ expresses Pax6, and the more ventral VZ expresses Nkx2.2. Properties of neuronal precursors located in the VZ are determined by the characteristic expression patterns of these transcription factors, leading to the generation of distinct classes of neurons. Recent studies demonstrated that radial glial cells produce neurons in addition to glia during central nervous system development. Thus, neuronal precursor diversity may be dependent upon the diversity of radial glial cells. To investigate this hypothesis, we analyzed the expression of radial glial cell markers and transcription factors in the mouse embryonic spinal cord. We show that radial glial cells indeed express domain-specific transcription factor. Moreover, they varied in expression of the astrocyte-specific glutamate transporter. The region where the astrocyte-specific glutamate transporter is strongly expressed in the ventral radial glial cells is closely related to the Pax6-expressing domain, and the weakly expressing region corresponding to the Nkx2.2-expressing domain. Furthermore, dorsal radial fibers expressed ephrin-B1. Thus, different types of radial glial cells exist in different domains defined by the transcription factor expression at E12.5. We also show that this diversity continues to the gliogenic stage of radial glial cells. This raises the idea that astrocytes generated from different domains along the dorsoventral axis in the mouse spinal cord have distinct characteristics. Copyright (c) 2005 S. Karger AG, Basel. PMID: 16280634 [PubMed - in process] --------------------------------------------------------------- 2: Int J Dev Biol. 2005;49(2-3):231-5. Isthmus organizer and regionalization of the mesencephalon and metencephalon. Nakamura H, Watanabe Y. Department of Molecular Neurobiology, Graduate School of Life Sciences and Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan. nakamura@idac.tohoku.ac.jp The brain vesicles that are formed at an early stage of neural development are the fundamentals of the brain plan. Heterotopic transplantation revealed that the diencephalon could change its fate when juxtaposed to the isthmus (mes-metencephalic boundary), which indicated that the isthmus functions as an organizer for the mesencephalon and metencephalon. Fgf8 is identified as an isthmus organizing signal. Misexpression of Fgf8a and Fgf8b indicated that a strong Fgf8 signal organizes cerebellar development. The transcription factors define the fate of the region. Overlapping expression of Otx2, En1 and Pax2 may define the mesencephalic region and additional expression of Pax3/7 may instruct the mesencephalic region to differentiate into the tectum. The di-mesencephalic boundary is determined by repressive interaction between Pax6 and En1/Pax2 and the mes-metencephalic boundary is defined by repressive interaction between Otx2 and Gbx2. Fgf8 is induced at the border of the Otx2 and Gbx2 expression domain, overlapping with Gbx2 expression. Publication Types: Review Review, Tutorial PMID: 15906236 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Dev Dyn. 2005 Feb;232(2):385-92. Potential roles for BMP and Pax genes in the development of iris smooth muscle. Jensen AM. Biology Department, 221 Morrill South, University of Massachusetts, Amherst, MA 01003, USA. ajensen@bio.umass.edu The embryonic optic cup generates four types of tissue: neural retina, pigmented epithelium, ciliary epithelium, and iris smooth muscle. Remarkably little attention has focused on the development of the iris smooth muscle since Lewis ([1903] J. Am. Anat. 2:405-416) described its origins from the anterior rim of the optic cup neuroepithelium. As an initial step toward understanding iris smooth muscle development, I first determined the spatial and temporal pattern of the development of the iris smooth muscle in the chick by using the HNK1 antibody, which labels developing iris smooth muscle. HNK1 labeling shows that iris smooth muscle development is correlated in time and space with the development of the ciliary epithelial folds. Second, because neural crest is the only other neural tissue that has been shown to generate smooth muscle (Le Lievre and Le Douarin [1975] J. Embryo. Exp. Morphol. 34:125-154), I sought to determine whether iris smooth muscle development shares similarities with neural crest development. Two members of the BMP superfamily, BMP4 and BMP7, which may regulate neural crest development, are highly expressed by cells at the site of iris smooth muscle generation. Third, because humans and mice that are heterozygous for Pax6 mutations have no irides (Hill et al. [1991] Nature 354:522-525; Hanson et al. [1994] Nat. Genet. 6:168-173), I determined the expression of Pax6. I also examined the expression of Pax3 in the developing anterior optic cup. The developing iris smooth muscle coexpresses Pax6 and Pax3. I suggest that some of the eye defects caused by mutations in Pax6, BMP4, and BMP7 may be due to abnormal iris smooth muscle. Copyright 2004 Wiley-Liss, Inc. PMID: 15614784 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Cell Cycle. 2004 Dec;3(12):1645-50. Epub 2004 Dec 4. A dynamic switch in the replication timing of key regulator genes in embryonic stem cells upon neural induction. Perry P, Sauer S, Billon N, Richardson WD, Spivakov M, Warnes G, Livesey FJ, Merkenschlager M, Fisher AG, Azuara V. Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, UK. Mammalian embryonic stem (ES) cells can either self-renew or generate progenitor cells that have a more restricted developmental potential. This provides an important model system to ask how pluripotency, cell commitment and differentiation are regulated at the level of chromatin-based changes that distinguish stem cells from their differentiated progeny. Here we show that the differentiation of ES cells to neural progenitors results in dynamic changes in the epigenetic status of multiple genes that encode transcription factors critical for early embryonic development or lineage specification. In particular, we demonstrate that DNA replication at a subset of neural-associated genes including Pax3, Pax6, Irx3, Nkx2.9 and Mash1 is advanced upon neural induction, consistent with increased locus accessibility. Conversely, many ES-associated genes including Oct4, Nanog, Utf1, Foxd3, Cripto and Rex1 that replicate early in ES cells switch their replication timing to later in S-phase in response to differentiation. Detailed analysis of the Rex1 locus reveals that delayed replication extends to a 2.8 Mb region surrounding the gene and is associated with substantial reductions in the level of histone H3K9 and H4 acetylation at the promoter. These results show that loss of pluripotency (and lineage choice) is associated with extensive and predictable changes in the replication timing of key regulator genes. PMID: 15611653 [PubMed - in process] --------------------------------------------------------------- 5: Int J Dev Neurosci. 2004 Jun;22(4):215-24. MyoD-lacZ transgenes are early markers in the neural retina, but MyoD function appears to be inhibited in the developing retinal cells. Kablar B. Department of Anatomy and Neurobiology, Dalhousie University, 5850 College Street, Halifax, NS, Canada B3H 5X1. bkablar@dal.ca Recent findings suggest that eye and skeletal muscle development in vertebrates share the same regulatory network. In that network, Pax3 gene is apparently activated through Dach/Eya/Six feedback loop to mediate MyoD-driven myogenesis. The purpose of this study was to investigate previously reported MyoD-lacZ expression in the developing mouse neural retina and to gain insight into the potential role of MyoD in the embryonic retinal cells. The analysis of MD6.0-lacZ and 258/-2.5lacZ transgenic embryos revealed that the retinal temporal expression pattern of the two transgenes resembled their expression pattern in the MyoD-dependent precursor muscle cells. However, MyoD transcripts and protein could not be found in the sites of MyoD-lacZ retinal expression. Furthermore, our immunohistochemical analysis suggests the existence of diverse factors (e.g., Pax6 and Chx10) within the retinal cells that differentially and inappropriately activate the two transgenes. Finally, the retinal phenotype observed in Pax7-/- knock-out mice suggests a role for Pax7 in photoreceptor cell differentiation, retinal lamination and in the etiopathology of retinoblastoma. Taken together, our data suggest that the MyoD gene evolved a different mechanism to achieve its down-regulation within the retina than that of the Myf5 gene. PMID: 15245757 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Dev Biol. 2004 Jul 15;271(2):439-66. Molecular anatomy of placode development in Xenopus laevis. Schlosser G, Ahrens K. Brain Research Institute, University of Bremen, 28334 Bremen, Germany. gschloss@uni-bremen.de We analyzed the spatiotemporal pattern of expression of 15 transcription factors (Six1, Six4, Eya1, Sox3, Sox2, Pax6, Pax3, Pax2, Pax8, Dlx3, Msx1, FoxI1c, Tbx2, Tbx3, Xiro1) during placode development in Xenopus laevis from neural plate to late tail bud stages. Out of all genes investigated, only the expression of Eya1, Six1, and Six4 is maintained in all types of placode (except the lens) throughout embryonic development, suggesting that they may promote generic placodal properties and that their crescent-shaped expression domain surrounding the neural plate defines a panplacodal primordium from which all types of placode originate. Double-labeling procedures were employed to reveal the precise position of this panplacodal primordium relative to neural plate, neural crest, and other placodal markers. Already at neural plate stages, the panplacodal primordium is subdivided into several subregions defined by particular combinations of transcription factors allowing us to identify the approximate regions of origin of various types of placode. Whereas some types of placode were already prefigured by molecularly distinct areas at neural plate stages, the epibranchial, otic, and lateral line placodes arise from a common posterior placodal area (characterized by Pax8 and Pax2 expression) and acquire differential molecular signatures only after neural tube closure. Our findings argue for a multistep mechanism of placode induction, support a combinatorial model of placode specification, and suggest that different placodes evolved from a common placodal primordium by successive recruitment of new inducers and target genes. PMID: 15223346 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Mol Cell Biol. 2004 Jun;24(11):4824-34. Paired-type homeodomain transcription factors are imported into the nucleus by karyopherin 13. Ploski JE, Shamsher MK, Radu A. The Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Box 1496, The Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. Jonathan.Ploski@mssm.edu We report that the paired homeodomain transcription factor Pax6 is imported into the nucleus by the Karyopherin beta family member Karyopherin 13 (Kap13). Pax6 was identified as a potential cargo for Kap13 by a yeast two-hybrid screen. Direct binding of Pax6 to Kap13 was subsequently confirmed by in vitro assays with recombinant proteins, and binding in vivo was shown by coimmunoprecipitation. Ran-dependent import of Pax6 by Kap13 was shown to occur by using a digitonin-permeabilized cells assay. Kap13 binds to Pax6 via a nuclear localization sequence (NLS), which is located within a segment of 80 amino acid residues that includes the homeodomain. Kap13 showed reduced binding to Pax6 when either region located at each end of the homeodomain (208 to 214 and 261 to 267) was deleted. The paired-type homeodomain transcription factor family includes more than 20 members. All members contain a region similar to the NLS found in Pax6 and are therefore likely to be imported by Kap13. We confirmed this hypothesis for Pax3 and Crx, which bind to and are imported by Kap13. PMID: 15143176 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Klin Oczna. 2003;105(3-4):207-10. [Formation of congenital defects during ocular development. II. Genetic background of oculogenesis and developmental birth defects] [Article in Polish] Krawczynski MR. Pracowni Poradnictwa Genetycznego w Chorobach Narzadu Wzroku Katedry, Zakladu Genetyki Medycznej Akademii Medycznej w Poznaniu. The course of oculogenesis is genetically controlled by genes, that are selectively expressed in the developing eye. Many of them, first recognized in studies on model animals, such as fruit fly (Drosophila melanogaster) proved, that they play similar roles in humans. The crucial role in cascade of genetic control of oculogenesis plays a system of genes PAX6-EYA1/SIX3-DACH1. In this work there are described also other genes, that form a network of interactions and regulations, such as: BMP4, BMP7, OPTX2, SOX1, PAX2, PITX2, PAX3, CHX10 and other genes. Describing function of each gene, author presented simultaneously all known congenital defects and syndromes, that are caused by mutations of above mentioned genes, such as: aniridia, anophthalmia, Peters anomaly and others. Publication Types: Review Review, Tutorial PMID: 14552188 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Oncogene. 2003 Sep 11;22(39):7989-97. Paired-Box genes are frequently expressed in cancer and often required for cancer cell survival. Muratovska A, Zhou C, He S, Goodyer P, Eccles MR. Department of Pathology, Dunedin School of Medicine, University of Otago, PO Box 913, Dunedin, New Zealand. The paired-box (PAX) genes encode a family of nine well-characterized paired-box transcription factors, with important roles in development and disease. Although PAX genes are primarily expressed in the embryo, constitutive expression promotes tissue hyperplasia. Rare tumor-specific mutations of PAX genes implicate an oncogenic role, and persistent PAX expression characterizes several tumors. Yet, a cancer-wide analysis of PAX gene expression to investigate a general role for PAX genes has not been performed. We analysed the pattern and requirement for PAX gene expression in a panel of common cancer cell lines. Very frequent PAX gene expression was identified in tumor cell lines, including lymphoma, breast, ovarian, lung, and colon cancer. In addition, the PAX2 gene was frequently expressed in a panel of 406 common primary tumor tissues. Apoptosis was rapidly induced in ovarian and bladder cancer cell lines following RNA interference to silence PAX2 expression, despite concomitant TP53 and/or HRAS mutations. These data suggest that PAX genes are frequently expressed in cancer, and that endogenous PAX gene expression is required for the growth and survival of cancer cells. PMID: 12970747 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Mech Dev. 2001 Apr;102(1-2):169-79. Characterization of mouse Dach2, a homologue of Drosophila dachshund. Davis RJ, Shen W, Sandler YI, Heanue TA, Mardon G. Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. The Drosophila genes eyeless, eyes absent, sine oculis and dachshund cooperate as components of a network to control retinal determination. Vertebrate homologues of these genes have been identified and implicated in the control of cell fate. We present the cloning and characterization of mouse Dach2, a homologue of dachshund. In situ hybridization studies demonstrate Dach2 expression in embryonic nervous tissues, sensory organs and limbs. This pattern is similar to mouse Dach1, suggesting a partially redundant role for these genes during development. In addition, we determine that Dach2 expression in the forebrain of Pax6 mutants and dermamyotome of Pax3 mutants is not detectably altered. Finally, genetic mapping experiments place mouse Dach2 on the X chromosome between Xist and Esx1. The identification of human DACH2 sequences at Xq21 suggests a possible role for this gene in Allan-Herndon syndrome, Miles-Carpenter syndrome, X-linked cleft palate and/or Megalocornea. PMID: 11287190 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Biochem Cell Biol. 2000;78(5):629-38. Genetic and biochemical diversity in the Pax gene family. Underhill DA. Department of Medical Genetics, University of Alberta, Edmonton, Canada. alan.underhill@ualberta.ca The mammalian Pax gene family comprises nine members that are characterized by a conserved DNA-binding motif, the paired domain, which was originally described in the Drosophila protein paired. Both loss- and gain-of-function studies reveal that Pax genes carry out essential roles during embryogenesis, and in some instances, may function as master regulatory genes. This review focuses on both genetic and biochemical aspects of the Pax family, and emphasizes important differences in the activity of individual Pax genes and their protein products. Publication Types: Review Review, Tutorial PMID: 11103953 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Genes Dev. 1999 Dec 15;13(24):3231-43. Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation. Heanue TA, Reshef R, Davis RJ, Mardon G, Oliver G, Tomarev S, Lassar AB, Tabin CJ. Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 USA. We have identified a novel vertebrate homolog of the Drosophila gene dachshund, Dachshund2 (Dach2). Dach2 is expressed in the developing somite prior to any myogenic genes with an expression profile similar to Pax3, a gene previously shown to induce muscle differentiation. Pax3 and Dach2 participate in a positive regulatory feedback loop, analogous to a feedback loop that exists in Drosophila between the Pax gene eyeless (a Pax6 homolog) and the Drosophila dachshund gene. Although Dach2 alone is unable to induce myogenesis, Dach2 can synergize with Eya2 (a vertebrate homolog of the Drosophila gene eyes absent) to regulate myogenic differentiation. Moreover, Eya2 can also synergize with Six1 (a vertebrate homolog of the Drosophila gene sine oculis) to regulate myogenesis. This synergistic regulation of muscle development by Dach2 with Eya2 and Eya2 with Six1 parallels the synergistic regulation of Drosophila eye formation by dachshund with eyes absent and eyes absent with sine oculis. This synergistic regulation is explained by direct physical interactions between Dach2 and Eya2, and Eya2 and Six1 proteins, analogous to interactions observed between the Drosophila proteins. This study reveals a new layer of regulation in the process of myogenic specification in the somites. Moreover, we show that the Pax, Dach, Eya, and Six genetic network has been conserved across species. However, this genetic network has been used in a novel developmental context, myogenesis rather than eye development, and has been expanded to include gene family members that are not directly homologous, for example Pax3 instead of Pax6. PMID: 10617572 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Int J Dev Biol. 1999 Sep;43(6):501-8. Expression pattern of PAX3 and PAX6 genes during human embryogenesis. Terzic J, Saraga-Babic M. Department of Physiology, Medical School, University of Split, Croatia. The expression of human PAX6 and PAX3 genes was investigated in 6 human 6-9 week old conceptuses by in situ hybridization. During human embryonic development (6-8 developmental weeks), PAX6 was expressed in the ventricular zone of telencephalon and diencephalon, and in the ventricular and ventral intermediate zones of medulla oblongata and spinal cord. PAX6 expression was detected in both layers of the optic cup, optic stalk and prospective corneal epithelium. Infundibulum and Rathke's pouch of the pituitary gland showed hybridization signal as well. In the early fetal period (9 developmental weeks) PAX6 expression increased in the spinal cord. In the eye, hybridization signal characterized the corneal and lens epithelium, pigmented and neural retina, while it was missing in the optic disc and nerve. In a 6-week human embryo, transcripts of PAX3 gene were observed in the ventricular zone at the mesencephalic-rhombencephalic border, and in the dorsal part of the ventricular zone and the roof plate of the medulla oblongata and the spinal cord. In the 8-9-week fetus, PAX3 expression increased in dorsal parts of the spinal cord. PAX3 characterized ectomesenchyme of the upper and lower jaw, and tongue. During early human development, PAX6 and PAX3 genes seem to be involved in the brain regionalization and establishment of dorso-ventral polarity of the spinal cord. Additionally, PAX6 participates in organogenesis of the eye and the pituitary gland, and PAX3 in the development of face and neck mesenchyme. PMID: 10610023 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Cell Mol Biol (Noisy-le-grand). 1999 Jul;45(5):601-16. The role of the notochord for epaxial myotome formation in the mouse. Dietrich S, Schubert FR, Gruss P, Lumsden A. King's College London, Department of Craniofacial Development, UK. s.dietrich@umds.ac.uk The vertebrate somite is the source of all trunk skeletal muscles. Myogenesis in avian embryos is thought to depend on signals from notochord and neural tube for the epaxial muscles, and signals from lateral mesoderm and surface ectoderm for the hypaxial muscles. However, this hypothesis has to be tested because in mouse mutants lacking a notochord the presence of a fused myotome beneath the neural tube has been reported. We have compared the expression pattern of myogenic markers and markers for the hypaxial muscle precursors in the mutants Brachyury curtailed, truncate, Danforth's short tail and Pintail. In regions lacking notochord and sclerotome, we found small, ventrally located domains of Myf5 and MyoD expression, concomitant with ventrally expanded Pax3 signals and upregulated expression of the hypaxial marker Lbx1, suggesting that only the hypaxial program is active. We therefore hypothesise that in mammals, as in birds, the formation of the epaxial musculature depends on the presence of notochord derived signals. PMID: 10512192 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Mech Dev. 1999 Jun;84(1-2):17-30. Retinoic acid induces down-regulation of Wnt-3a, apoptosis and diversion of tail bud cells to a neural fate in the mouse embryo. Shum AS, Poon LL, Tang WW, Koide T, Chan BW, Leung YC, Shiroishi T, Copp AJ. Department of Anatomy, The Chinese University of Hong Kong, Shatin. alisa-shum@cuhk.edu.hk The tail bud comprises the caudal extremity of the vertebrate embryo, containing a pool of pluripotent mesenchymal stem cells that gives rise to almost all the tissues of the sacro-caudal region. Treatment of pregnant mice with 100 mg/kg all-trans retinoic acid at 9.5 days post coitum induces severe truncation of the body axis, providing a model system for studying the mechanisms underlying development of caudal agenesis. In the present study, we find that retinoic acid treatment causes extensive apoptosis of tail bud cells 24 h after treatment. Once the apoptotic cells have been removed, the remaining mesenchymal cells differentiate into an extensive network of ectopic tubules, radially arranged around the notochord. These tubules express Pax-3 and Pax-6 in a regionally-restricted pattern that closely resembles expression in the definitive neural tube. Neurofilament-positive neurons subsequently grow out from the ectopic tubules. Thus, the tail bud cells remaining after retinoic acid-induced apoptosis appear to adopt a neural fate. Wnt-3a, a gene that has been shown to be essential for tail bud formation, is specifically down-regulated in the tail bud of retinoic acid-treated embryos, as early as 2 h after retinoic acid treatment and Wnt-3a transcripts become undetectable by 10 h. In contrast, Wnt-5a and RAR-gamma are still detectable in the tail bud at that time. Extensive cell death also occurs in the tail bud of embryos homozygous for the vestigial tail mutation, in which there is a marked reduction in Wnt-3a expression. These embryos go on to develop multiple neural tubes in their truncated caudal region. These results suggest that retinoic acid induces down-regulation of Wnt-3a which may play an important role in the pathogenesis of axial truncation, involving induction of widespread apoptosis, followed by an alteration of tail bud cell fate to form multiple ectopic neural tubes. PMID: 10473117 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2420-5. A binding site for homeodomain and Pax proteins is necessary for L1 cell adhesion molecule gene expression by Pax-6 and bone morphogenetic proteins. Meech R, Kallunki P, Edelman GM, Jones FS. Department of Neurobiology, The Scripps Research Institute and the Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. The cell adhesion molecule L1 regulates axonal guidance and fasciculation during development. We previously identified the regulatory region of the L1 gene and showed that it was sufficient for establishing the neural pattern of L1 expression in transgenic mice. In the present study, we characterize a DNA element within this region called the HPD that 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 was necessary for binding to Pax-6. In cellular transfection experiments, L1-luciferase reporter constructs containing the HPD were 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 were eliminated on deletion of the HPD from L1 constructs. In transgenic mice, deletion of the HPD from an L1-lacZ reporter resulted in a loss of beta-galactosidase expression in the telencephalon and mesencephalon. Collectively, our 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. PMID: 10051657 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: J Cell Sci. 1999 Jan;112 ( Pt 2):253-61. Induction of antisense Pax-3 expression leads to the rapid morphological differentiation of neuronal cells and an altered response to the mitogenic growth factor bFGF. Reeves FC, Burdge GC, Fredericks WJ, Rauscher FJ, Lillycrop KA. Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, UK. KAL@soton.ac.uk. Mutations within the Pax-3 gene lead to a range of developmental abnormalities in both humans and mice. In this report, we have investigated the role that Pax-3 plays in neuronal cell development by specifically downregulating Pax-3 expression within a neuronal cell line. This was achieved by stably transfecting the neuronal cell line ND7 with an expression vector in which antisense Pax-3 RNA was produced under the control of the inducible MMTV promoter. In the stable transfectants, we found that the addition of dexamethasone led to the induction of antisense Pax-3 RNA and a rapid downregulation in endogenous Pax-3 protein expression. The decrease in endogenous Pax-3 protein expression corresponded with a dramatic change in the morphology of the cell: the normally rounded ND7 cells exhibited increased cell to substrate adhesion, extended long neurite processes and expressed genes such as snap-25 that are characteristic of a mature neuron. The morphological differentiation induced by a reduction in Pax-3 expression was followed 24-48 hours later by a cessation in cell proliferation. Interestingly the morphological differentiation and cessation in cell proliferation inducted in the cell lines lacking Pax-3 could be reversed by the addition of the mitogenic growth factor EGF but not by bFGF, whose receptor was downregulated in these cells. These results suggest that the expression of Pax-3 is essential to maintain the undifferentiated phenotype of these immature neuronal cells, and in its absence the cells acquire many of the characteristics of a mature neuronal cell. The slow onset of cell cycle arrest in the cells lacking Pax-3 argues against this transcription factor playing a direct role in the regulation of neuronal cell proliferation. PMID: 9858478 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Mech Dev. 1998 Apr;73(1):59-72. Abnormalities of floor plate, notochord and somite differentiation in the loop-tail (Lp) mouse: a model of severe neural tube defects. Greene ND, Gerrelli D, Van Straaten HW, Copp AJ. Neural Development Unit, Institute of Child Health, University College, 30 Guilford Street, London, WC1N 1EH, UK. Mouse embryos homozygous for the loop-tail (Lp) mutation fail to initiate neural tube closure at E8.5, leading to a severe malformation in which the neural tube remains open from midbrain to tail. During initiation of closure, the normal mouse neural plate bends sharply in the midline, at the site of the future floor plate. In contrast, Lp/Lp embryos exhibit a broad region of flat neural plate in the midline, displacing the sites of neuroepithelial bending to more lateral positions. Sonic hedgehog (Shh) and Netrin1 are expressed in abnormally broad domains in the ventral midline of the E9.5 Lp/Lp neural tube, suggesting over-abundant differentiation of the floor plate. The notochord is also abnormally broad in Lp/Lp embryos with enlarged domains of Shh and Brachyury expression. The paraxial mesoderm shows evidence of ventralisation, with increased expression of the sclerotomal marker Pax1, and diminished expression of the dermomyotomal marker Pax3. While the expression domain of Pax3 does not differ markedly from wild-type, there is a dorsal shift in the domain of Pax6 expression in the neural tube at caudal levels of Lp/Lp embryos. We suggest that the Lp mutation causes excessive differentiation of floor-plate and notochord, with over-production of Shh from these midline structures causing ventralisation of the paraxial mesoderm and, to a lesser extent, the neural tube. Comparison with other mouse mutants suggests that the enlarged floor plate may be responsible for the failure of neural tube closure in Lp/Lp embryos. Copyright 1998 Elsevier Science Ireland Ltd. PMID: 9545534 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: J Biol Chem. 1997 Nov 7;272(45):28289-95. The C-terminal subdomain makes an important contribution to the DNA binding activity of the Pax-3 paired domain. Vogan KJ, Gros P. Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada. The recognition of DNA targets by Pax-3 is achieved through the coordinate use of two distinct helix-turn-helix-based DNA-binding modules: a paired domain, composed of two structurally independent subdomains joined by a short linker, and a paired-type homeodomain. In mouse, the activity of the Pax-3 paired domain is modulated by an alternative splicing event in the paired domain linker region that generates isoforms (Q+ and Q-) with distinct C-terminal subdomain-mediated DNA-binding properties. In this study, we have used derivatives of a classical high affinity paired domain binding site (CD19-2/A) to derive an improved consensus recognition sequence for the Pax-3 C-terminal subdomain. This new consensus differs at six out of eight positions from the C-terminal subdomain recognition motif present in the parent CD19-2/A sequence, and includes a 5'-TT-3' dinucleotide at base pairs 15 and 16 that promotes high affinity binding by both Pax-3 isoforms. However, with a less favorable guanine at position 15, only the Q- isoform retains high affinity binding to this sequence, suggesting that this alternative splicing event might serve to stabilize binding to suboptimal recognition sequences. Finally, mutagenic analysis of the linker demonstrates that both the sequence and the spacing in this region contribute to the enhanced DNA-binding properties of the Pax-3/Q- isoform. Altogether, our studies establish a clear role for the Pax-3 C-terminal subdomain in DNA recognition and, thus, provide insights into an important mechanism by which Pax proteins achieve distinct target specificities. PMID: 9353283 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Bioessays. 1997 Sep;19(9):755-65. Pax genes and organogenesis. Dahl E, Koseki H, Balling R. GSF-National Research Center for Environment and Health, Institute for Mammalian Genetics, Neuherberg, Germany. Pax genes are a family of developmental control genes that encode nuclear transcription factors. They are characterized by the presence of the paired domain, a conserved amino acid motif with DNA-binding activity. Originally, paired-box-containing genes were detected in Drosophila melanogaster, where they exert multiple functions during embryogenesis. In vertebrates, Pax genes are also involved in embryogenesis. Mutations in four out of nine characterized Pax genes have been associated with either congenital human diseases such as Waardenburg syndrome (PAX3), Aniridia (PAX6), Peter's anomaly (PAX6), renal coloboma syndrome (PAX2) or spontaneous mouse mutants (undulated (Pax1), Splotch (Pax3), Small eye (Pax6), Pax2(1)Neu), which all show defects in development. Recently, analysis of spontaneous and transgenic mouse mutants has revealed that vertebrate pax genes are key regulators during organogenesis of kidney, eye, ear, nose, limb muscles, vertebral column and brain. Like their Drosophila counterparts, vertebrate Pax genes are involved in pattern formation during embryogenesis, possibly by determining the time and place of organ initiation or morphogenesis. For most tissues, however, the nature of the primary developmental action of Pax transcription factors remains to be elucidated. One predominant theme is signal transduction during tissue interactions, which may lead to a position-specific regulation of cell proliferation. Publication Types: Review Review, Tutorial PMID: 9297966 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Development. 1997 Jun;124(12):2335-44. Dorsoventral patterning of the vertebrate neural tube is conserved in a protochordate. Corbo JC, Erives A, Di Gregorio A, Chang A, Levine M. Department of Molecular and Cellular Biology, University of California, Berkeley 94720, USA. The notochord and dorsal ectoderm induce dorsoventral compartmentalization of the vertebrate neural tube through the differential regulation of genes such as HNF-3beta, Pax3, Pax6 and snail. Here we analyze the expression of HNF-3beta and snail homologues in the ascidian, Ciona intestinalis, a member of the subphylum Urochordata, the earliest branch in the chordate phylum. A combination of in situ hybridization and promoter fusion analyses was used to demonstrate that the Ciona HNF-3beta homologue is expressed in the ventralmost ependymal cells of the neural tube, while the Ciona snail homologue is expressed at the junction between the invaginating neuroepithelium and dorsal ectoderm, similar to the patterns seen in vertebrates. These findings provide evidence that dorsoventral compartmentalization of the chordate neural tube is not an innovation of the vertebrates. We propose that precursors of the floor plate and neural crest were present in a common ancestor of both vertebrates and ascidians. PMID: 9199360 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Development. 1997 May;124(10):1985-97. Pax-6 functions in boundary formation and axon guidance in the embryonic mouse forebrain. Mastick GS, Davis NM, Andrew GL, Easter SS Jr. Department of Biology, University of Michigan, Ann Arbor 48109, USA. grants@umich.edu The Pax-6 gene encodes a transcription factor that is expressed in regionally restricted patterns in the developing brain and eye. Here we describe Pax-6 expression in the early forebrain (prosencephalon) on embryonic day 9.5 (E9.5) to E10.5 using both whole-mount in situ hybridization and antibody labeling. We find close correlations between Pax-6+ domains and initial neural patterning, and identify corresponding defects in embryos homozygous for the Pax-6 allele, Small eye (Sey). Pax-6 expression defines the prosencephalon-mesencephalon boundary, and mutant embryos lack this morphological boundary. Markers of the caudal prosencephalon are lost (Pax-6, Lim-1, Gsh-1) and a marker for mesencephalon is expanded rostrally into the prosencephalon (Dbx). We conclude that the caudal prosencephalon (prosomere 1) is at least partially transformed to a mesencephalic fate. This transformation results in a specific deficit of posterior commissure axons. Sey/Sey embryos also exhibit an axon pathfinding defect specific to the first longitudinal tract in the prosencephalon (tpoc, tract of the postoptic commissure). In wild type, tpoc axons fan out upon coming in contact with a superficial patch of Pax-6+ neuron cell bodies. In the mutant, the tpoc axons have normal initial projections, but make dramatic errors where they contact the neuron cell bodies, and fail to pioneer this first tract. Thus Pax-6 is required for local navigational information used by axons passing through its domain of expression. We conclude that Pax-6 plays multiple roles in forebrain patterning, including boundary formation, regional patterning, neuron specification and axon guidance. PMID: 9169845 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Dev Biol. 1997 Mar 15;183(2):150-65. A role for midline closure in the reestablishment of dorsoventral pattern following dorsal hindbrain ablation. Buxton P, Hunt P, Ferretti P, Thorogood P. Developmental Biology Unit, Institute of Child Health (University College London), Britain. The cellular and molecular study of dorsal neural tube ablation reported here demonstrates a critical role for midline closure in hindbrain repatterning. This was revealed by detailed analysis of the transcriptional response of two genes, Pax-3 and slug, during repair of the neural tube following ablation. The reexpression of Pax-3 appears to rely on a single surface ectoderm/neuroepithelial contact, while this is insufficient for reexpression of slug. In fact, slug up-regulation only occurred upon midline closure and, strikingly, corresponded to down-regulation of Pax-3. We examined whether a candidate dorsalizing molecule, Bmp-4, was responsible for this reciprocal regulation of Pax-3 and slug at midline closure. However, Bmp-4 was not reexpressed following ablation, indicating not only that it is not responsible for the observed repatterning but that it lies in regulatory pathways distinct from Pax-3 and slug. We additionally examined the expression of Pax-6, which, together with assessment of the pattern of cranial ganglia, roof plate morphology, and positioning of branchiomotor exit points, demonstrates that neural crest regeneration is accompanied by reestablishment of a normal dorsoventral pattern within the neural tube. Thus, both local and longer range patterning appears to be restored following ablation, which is reliant dorsally on midline closure of the neural tube. PMID: 9126291 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Mol Cell Biol. 1996 Dec;16(12):6677-86. An alternative splicing event in the Pax-3 paired domain identifies the linker region as a key determinant of paired domain DNA-binding activity. Vogan KJ, Underhill DA, Gros P. Department of Biochemistry, McGill University, Montreal, Quebec, Canada. We have identified alternatively spliced isoforms of murine Pax-3 and Pax-7 which differ by the presence or absence of a single glutamine residue in a linker region which separates two distinct DNA-binding subdomains within the paired domain. By reverse transcription-PCR, these isoforms of Pax-3 and Pax-7 (Q+ and Q-) were detected at similar levels through multiple developmental stages in the early mouse embryo. DNA-binding studies using the Q+ and Q- isoforms of Pax-3 revealed that this alternative splicing event had no major effect on the ability of these isoforms to bind to an oligonucleotide specific for the Pax-3 homeodomain (P2) or to a paired domain recognition sequence (e5) that interacts primarily with the N-terminal subdomain of the paired domain. However, DNA-binding studies with sequences (P6CON and CD19-2/A) containing consensus elements for both the N-terminal and C-terminal subdomains revealed that the Q- isoform binds to these sequences with a two- to fivefold-higher affinity; further mutation of the GTCAC core N-terminal subdomain recognition motif of CD19-2/A generated binding sites with a high degree of specificity for the Q- isoform. These differences in DNA binding in vitro were also reflected in the enhanced ability of the Q- isoform to stimulate transcription of a reporter containing multiple copies of CD19-2/A upstream of the thymidine kinase basal promoter. In support of the observations made with these naturally occurring Pax-3 isoforms, introducing a glutamine residue at the analogous position in PAX6 caused a fivefold reduction in binding to P6CON and a complete loss of binding to CD19-2/A and to the C-terminal subdomain-specific probe 5aCON. These studies therefore provide direct evidence for a role for the paired-domain linker region in DNA target site selection, and they identify novel isoforms of Pax-3 and Pax-7 that have the potential to mediate distinct functions in the developing embryo. PMID: 8943322 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Curr Opin Genet Dev. 1996 Jun;6(3):334-42. Transcription factors in disease. Engelkamp D, van Heyningen V. MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK. dieter@hgu.mrc.ac.uk Mutations affecting several predominantly tissue-specific transcriptional regulators have recently been associated with disease phenotypes. Although the mutational spectrum is variable, many of the reported cases involve clear loss-of-function mutations-such as Waardenburg syndrome type 1, aniridia and Rubinstein-Taybi syndrome-suggesting that the genetic mechanism involved in disease is haplo-insufficiency. The high degree of dosage sensitivity often appears to affect only a subset of the tissues that express the gene. Position effects with cytogenetic rearrangements well outside the coding region have been implicated for four of the genes discussed: POU3F4, SOX9, PAX6, and GL13. Publication Types: Review Review, Tutorial PMID: 8791518 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Gene. 1995 Sep 11;162(2):267-70. Identification of DNA recognition sequences for the Pax3 paired domain. Chalepakis G, Gruss P. Max-Planck Institute for Biophysical Chemistry, Gottingen, Germany. The Pax gene family, encoding transcription factors, has been classified into four subfamilies according to their genomic organization, the sequences of the paired domains (PD) and the expression pattern. Pax1 and Pax9 constitute one subfamily, Pax2, Pax5 and Pax8 another, Pax3 and Pax7 another one and Pax4 and Pax6 the fourth subfamily. The PD exhibits DNA-binding activity, and is the most conserved functional motif in all Pax proteins. A high-resolution analysis of a PD structure has been performed [Xu et al., Cell 80 (1995) 639-650] and the DNA-binding characteristics of members of two Pax subfamilies (Pax2, Pax5 and Pax6) have been determined. Here, we have utilized a PCR-based selection approach to identify the DNA-binding sequences of the Pax3/PD, a member of a subfamily which has not yet been characterized. Comparison of the Pax3/PD-binding sequences with those of other PD proteins revealed both similarities and differences in the DNA-recognition sequence. This suggests that different Pax proteins can regulate the expression of the same target gene, but they can also regulate the expression of completely unrelated genes by binding to their DNA regulatory regions. PMID: 7557441 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6952-6. Activin A inhibits Pax-6 expression and perturbs cell differentiation in the developing spinal cord in vitro. Pituello F, Yamada G, Gruss P. Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, Gottingen, Germany. We have developed an in vitro model of the isolated chicken neural plate. Here we demonstrate that even in the absence of notochord, the neural plate rapidly develops a typical dorsoventral patterning. This observation suggests that the ventral cell types are specified or at least predetermined prior to notochord formation and that permissive conditions are sufficient for differentiation of ventral structures. Treatment of the neural plate with activin A extinguishes Pax-6 gene expression, whereas the dorsal markers Pax-3 and Pax-7 are still expressed. The absence of Pax-6 transcripts can be correlated with an impeded differentiation of the motor neurons, whereas the floor plate seems to be enlarged. We propose that the region-specific expression of Pax-6 in the spinal cord is under the control of activin-like molecules. PMID: 7624351 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: C R Acad Sci III. 1995 Jan;318(1):57-66. PAX-genes expression during human embryonic development, a preliminary report. Gerard M, Abitbol M, Delezoide AL, Dufier JL, Mallet J, Vekemans M. Laboratoire d'histologie-embryologie-cytogenetique, Universite Rene-Descartes (Paris-V), Faculte de medecine Necker, France. PAX-genes encode important transcriptional factors during embryogenesis. They are also involved in human diseases, Waardenburg syndrome, Aniridia and tumors. We report in the present paper a preliminary in situ hybridization study of PAX3-, PAX5- and PAX6-gene expression during human embryonic development. PAX3-gene is expressed in the neural groove before closure, and in the closed neural tube. Afterwards, its expression is observed in the mesencephalon, the rhombencephalon and the spinal cord. PAX5-gene expression is restricted to the mesencephalon-rhombencephalon boundary and the spinal cord. PAX6-gene is expressed early in the neural tube, just after its closure. Afterwards, its expression is observed in the forebrain, the rhombencephalon, the somites and the spinal cord. These patterns of expression are observed early during human embryonic development and are specific in time and space. This preliminary report shows the feasibility of in situ hybridization methodology for studying the expression of developmental genes during the early stages of human embryogenesis. It opens the way to study the pathogenesis of polymalformative syndromes and tumorigenesis. PMID: 7757805 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Curr Opin Genet Dev. 1994 Jun;4(3):427-38. PAX genes. Strachan T, Read AP. Department of Human Genetics, University of Newcastle upon Tyne, UK. PAX genes are developmental control genes that encode transcription factors containing a DNA-binding paired domain. Mutations in three of the nine mouse genes (Pax1, Pax3 and Pax6) and two of the nine human genes (PAX3 and PAX6) are known to cause developmental defects. These defects are caused by loss-of-function alleles; pathogenesis occurs as a result of a half dosage of the PAX gene product in particular cells. Gain-of-function mutations have been implicated in cancer. Publication Types: Review Review, Tutorial PMID: 7919921 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: J Cell Biol. 1994 Apr;125(2):417-25. Differential induction of Pax genes by NGF and BDNF in cerebellar primary cultures. Kioussi C, Gruss P. Abteilung fur Molekular Zellbiologie, Max-Planck Institut fur Biophysikalische Chemie, Gottingen, Germany. The Pax genes encode sequence-specific DNA binding transcription factors that are expressed in embryonic development of the nervous system. Primary neuronal cell cultures derived from the cerebellar cortex of embryonic day 14, newborn and 7-d old mice, were used to investigate the cell-type specific expression patterns of three members of the murine paired box containing gene family (Pax gene family), in vitro. Cell types which express Pax-2, Pax-3, and Pax-6 RNA in primary cultures correspond to those found in regions of the cerebellum which show RNA signals in sections of the developing mouse brain. To find mechanisms regulating Pax gene expression during cerebellar development, the differential regulation of Pax-2, Pax-3, and Pax-6 by NGF and BDNF, two structurally related neurotrophins, was studied in such primary cultures. Pax-2 and Pax-6 RNA increased slightly by 1 h and remained elevated throughout a 24-h treatment with BDNF and NGF. Pax-3 RNA was not detected in newborn cultures, but underwent a rapid (1 h) and transient (2 h) induction upon treatment with either BDNF or NGF. No response was seen with EGF or FGF. Cycloheximide treatment amplified Pax-3 induction and prolonged the signal. Thus, Pax-3 induction resembles that of the immediate-early gene c-fos, which transduces growth factor signals during the development of particular neuronal/glial cell types. The changes in Pax expression were inductive rather than trophic. PMID: 8163557 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Biochem Biophys Res Commun. 1994 Mar 15;199(2):552-63. Regulated expression of Brachyury(T), Nkx1.1 and Pax genes in embryoid bodies. Yamada G, Kioussi C, Schubert FR, Eto Y, Chowdhury K, Pituello F, Gruss P. Department of Molecular Cell Biology, Max Planck Institute of Biophysical Chemistry, Gottingen, Germany. Embryonic stem cells (ES) can be exploited to analyze in vitro mechanisms of cellular differentiation. We have utilized ES-derived embryoid body formation in an attempt to study cell types resulting from in vitro differentiation. To this end, a variety of molecular markers, preferably those which have been associated with regulatory events during mouse embryogenesis, was employed. Specifically, Brachyury (T), Pax-3 and Pax-6 genes as well as Nkx-1.1 were used. We could demonstrate that the expression of these genes in vitro was regulated by growth factors such as activin A or bFGF. Implications of these findings and the possible applications for identifying new genes are discussed. PMID: 7907867 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Princess Takamatsu Symp. 1994;24:323-37. Non-receptor tyrosine kinases in mammalian neurogenesis. Aizawa S, Yagi T, Furuta Y, Ikawa Y, Nada S, Nakagawa H, Okada M. Laboratory of Molecular Oncology Tsukuba Life Science Center, Ibaraki, Japan. Several members of the Src family of non-receptor tyrosine kinases are expressed at high levels in embryonic neural tissues as well as in adult brain. Relatively little has been known, however, about their roles in neural development. Attempts to clarify this by production of mutant mice have been unsuccessful because of gene redundancy. We earlier isolated a new cytoplasmic protein tyrosine kinase, Csk, and showed that it inactivates uniquely all members of non-receptor tyrosine kinases in vitro. Here, we have generated Csk-deficient mouse embryos and shown that Csk is indeed an indispensable negative regulator for all non-receptor tyrosine kinases in vivo, and that regulated activity of these kinases is essential for normal development of mice at the neural stage. The signaling pathway through Src-family kinases during neurulation is also discussed. Publication Types: Review Review, Tutorial PMID: 8983085 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------