1: Prog Neurobiol. 2001 Dec;65(5):473-88. Regionalisation and acquisition of polarity in the optic tectum. Nakamura H. Department of Molecular Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, 980-8575, Sendai, Japan. makamura@idac.tohoku.ac.jp The optic tectum differentiates from the alar plate of the mesencephalon and receives retinal fibres in a precise retinotopic manner. Here, mechanisms of tectum polarisation and regionalisation are reviewed. Misexpression of Pax2, Pax5 or En can change the fate of the presumptive diencephalon to that of the tectum. Ephrin A2 and A5 are expressed in a gradient in the tectum, caudal high and rostral low, and may play important roles in the formation of a precise retinotectal projection map. Retinal fibres that express receptors for these ligands, and which come from the temporal retina, are repulsed by the ligands and do not invade the caudal tectum. Both En1 and En2 can regulate posterior characteristics in the tectum by inducing ephrin A2 and A5. Transplantation experiments in chick have indicated that the mes/metencephalic boundary works as an organiser for the tectum and the cerebellum. Fgf8 is a candidate signalling molecule in the organiser. Pax2/5, En, and Fgf8 are in a positive feedback loop for their expression such that misexpression of one of these genes in the diencephalon turns on the feedback loop and can result in induction of an optic tectum. Otx2 and Gbx2 appear to repress each other's expression and contribute to defining the posterior border of the tectum. Misexpression of Otx2 in the metencephalon can change the fate of its alar plate to a tectum, and misexpression of Gbx2 in the mesencephalon can cause anterior shifting of the caudal limit of the tectum. The anterior border of the tectum may be determined as a result of repressive interactions between Pax6 and En1/Pax2. Along the dorsoventral axis of the mesencephalon, Shh contributes to ventralize the tissue; that is, Shh can change the fate of the presumptive tectum to that of the tegmentum that is the ventral structure. It is proposed that the brain vesicle that expresses Otx2, Pax2, and En1 may differentiate into the tectum. Publication Types: Review Review, Tutorial PMID: 11689282 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Int J Cancer. 2001 Aug 15;93(4):459-67. Overexpression of Pax5 is not sufficient for neoplastic transformation of mouse neuroectoderm. Steinbach JP, Kozmik Z, Pfeffer P, Aguzzi A. Institute of Neuropathology, University Hospital, Zurich, Switzerland. The developmental control genes of the Pax family are essential for brain development. Several Pax genes are also involved in chromosomal translocations causing malignancies in humans, and Pax5 expression is deregulated in medulloblastomas. We have investigated whether Pax5 can induce tumors in the developing mouse brain. Primary mouse embryonic neuroectodermal cells were retrovirally transduced with mouse Pax5 and transplanted into the brain of syngeneic host mice. No tumors developed in 36 transplants after one year, and there were no alterations in the differentiation pattern of the neural transplants. We then generated transgenic mice expressing human Pax5 under control of the Engrailed-2 promoter, which is expressed in the cerebellar external granule cell layer and in medulloblastomas. Sustained expression was achieved in the cerebellum of transgenic animals throughout lifetime. Expression levels were similar to those observed in human medulloblastomas. Again, cerebellar morphogenesis was undisturbed, and no tumors arose. These results strongly argue against a dominant transforming activity of PAX5 in NEC and in cerebellar granule cell precursors of mice, and underline the restricted tissue-specificity of PAX5 related oncogenesis. Copyright 2001 Wiley-Liss, Inc. PMID: 11477548 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Nucleic Acids Res. 2001 Apr 15;29(8):E42-2. Optimizing the detection of nascent transcripts by RNA fluorescence in situ hybridization. van Raamsdonk CD, Tilghman SM. Howard Hughes Medical Institute and Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. An unusual feature of the mammalian genome is the number of genes exhibiting monoallelic expression. Recently random monoallelic expression of autosomal genes has been reported for olfactory and Ly-49 NK receptor genes, as well as for Il-2, Il-4 and Pax5. RNA fluorescence in situ hybridization (FISH) has been exploited to monitor allelic expression by visualizing the number of sites of transcription in individual nuclei. However, the sensitivity of this technique is difficult to determine for a given gene. We show that by combining DNA and RNA FISH it is possible to control for the hybridization efficiency and the accessibility and visibility of fluorescent probes within the nucleus. PMID: 11292856 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Trends Neurosci. 2001 Jan;24(1):32-9. Regionalization of the optic tectum: combinations of gene expression that define the tectum. Nakamura H. Dept of Molecular Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, 980-8575, Sendai, Japan. nakamura@idac.tohoku.ac.jp The optic tectum differentiates from the alar plate of the mesencephalon. Here, the molecular mechanisms for differentiation of the tectum are reviewed. Mis-expression of Pax2, Pax5 or En can change the fate of the presumptive diencephalon to become the tectum. En, Fgf8, Pax2 and Pax5, exist in a positive feedback loop for their expression so that mis-expression of any of these genes acts on the feedback loop resulting in induction of the optic tectum in the diencephalon. Otx2 and Gbx2 can repress the expression of each other and contribute to the formation of the posterior border of the tectum. Mis-expression of Otx2 in the metencephalon changed the fate of its alar plate to the tectum. The anterior border of the tectum might be determined as a result of repressive interaction of Pax6 with En1 and Pax2. Along the dorsoventral axis of the mesencephalon, Shh contributes to the ventralization of the tissue, that is, the area affected by Shh differentiates into the tegmentum. It is proposed that the brain vesicle that expresses Otx2, Pax2 and En1 might differentiate into the tectum. Publication Types: Review PMID: 11163885 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Development. 2001 Jan;128(2):181-91. EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region. Liu A, Joyner AL. Howard Hughes Medical Institute and Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA. Fgf8, which is expressed at the embryonic mid/hindbrain junction, is required for and sufficient to induce the formation of midbrain and cerebellar structures. To address through what genetic pathways FGF8 acts, we examined the epistatic relationships of mid/hindbrain genes that respond to FGF8, using a novel mouse brain explant culture system. We found that En2 and Gbx2 are the first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain explants treated with FGF8-soaked beads. By examining gene expression in En1/2 double mutant mouse embryos, we found that Fgf8, Wnt1 and Pax5 do not require the En genes for initiation of expression, but do for their maintenance, and Pax6 expression is expanded caudally into the midbrain in the absence of EN function. Since E9.5 En1/2 double mutants lack the mid/hindbrain region, forebrain mutant explants were treated with FGF8 and, significantly, the EN transcription factors were found to be required for induction of Pax5. Thus, FGF8-regulated expression of Pax5 is dependent on EN proteins, and a factor other than FGF8 could be involved in initiating normal Pax5 expression in the mesencephalon/metencephalon. The En genes also play an important, but not absolute, role in repression of Pax6 in forebrain explants by FGF8. Previous Gbx2 gain-of-function studies have shown that misexpression of Gbx2 in the midbrain can lead to repression of Otx2. However, in the absence of Gbx2, FGF8 can nevertheless repress Otx2 expression in midbrain explants. In contrast, Wnt1 is initially broadly induced in Gbx2 mutant explants, as in wild-type explants, but not subsequently repressed in cells near FGF8 that normally express Gbx2. Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts downstream of FGF8 in repression of Wnt1. This is the first such epistatic study performed in mouse that combines gain-of-function and loss-of-function approaches to reveal aspects of mouse gene regulation in the mesencephalon/metencephalon that have been difficult to address using either approach alone. PMID: 11124114 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Development. 2000 Jun;127(11):2357-65. Pax6 defines the di-mesencephalic boundary by repressing En1 and Pax2. Matsunaga E, Araki I, Nakamura H. Department of Molecular Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan. Transcriptional factors and signaling molecules are responsible for regionalization of the central nervous system. In the early stage of neural development, Pax6 is expressed in the prosencephalon, while En1 and Pax2 are expressed in the mesencephalon. Here, we misexpressed Pax6 in the mesencephalon to elucidate the mechanism of the di-mesencephalic boundary formation. Histological analysis, expression patterns of diencephalic marker genes, and fiber trajectory of the posterior commissure indicated that Pax6 misexpression caused a caudal shift of the di-mesencephalic boundary. Pax6 repressed En1, Pax2 and other tectum (mesencephalon)-related genes such as En2, Pax5, Pax7, but induced Tcf4, a diencephalon marker gene. To know how Pax6 represses En1 and Pax2, we ectopically expressed a dominant-active or negative form of Pax6. The dominant-active form of Pax6 showed a similar but more severe phenotype than Pax6, while the dominant-negative form showed an opposite phenotype, suggesting that Pax6 acts as a transcriptional activator. Thus Pax6 may repress tectum-related genes by activating an intervening repressor. The results of misexpression experiments, together with normal expression patterns of Pax6, En1 and Pax2, suggest that repressive interaction between Pax6 and En1/Pax2 defines the di-mesencephalic boundary. PMID: 10804178 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Dev Biol. 2000 May 1;221(1):168-80. Antagonizing activity of chick Grg4 against tectum-organizing activity. Sugiyama S, Funahashi J, Nakamura H. Department of Molecular Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai, 980-8575, Japan. sugis@idac.tohoku.ac.jp Alar plate of chick mesencephalon differentiates into the optic tectum. It has been shown that factors expressed in the mes-metencephalic boundary induce the tectum and give positional specificity. Chick Grg4 is expressed at first in the anterior neural fold. The expression localizes from the posterior diencephalon to the mesencephalon by stage 10. To investigate the function of Grg4 in mesencephalic development, Grg4 overexpression was carried out by in ovo electroporation. After Grg4 overexpression, expression of En-2, Pax5, Fgf8, and EphrinA2 was repressed, and Pax6 was upregulated in the mesencephalic region. Grg4 overexpression caused the morphological change; mesencephalic swelling became smaller and the di-mesencephalic boundary shifted posteriorly, that is, the anterior limit of tectum shifted posteriorly. Importantly, cotransfection of Grg4 with Pax5 canceled the tectum-inducing activity of Pax5. These results suggest that Grg4 works as an antagonist against tectum-organizing activity. It was also shown that transfected N-terminal domains of Grg4 induced En-2 expression. Since N-terminal domains were transported to the nucleus in the neuroepithelium, they could act as dominant negative for endogenous Grg4. These results indicate that Grg4 has repressing activity against the organizing molecules and suggest that Grg4 plays important roles in formation of anterior tectal boundary and polarity. Copyright 2000 Academic Press. PMID: 10772799 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: 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] --------------------------------------------------------------- 9: Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5709-13. Deregulated expression of PAX5 in medulloblastoma. Kozmik Z, Sure U, Ruedi D, Busslinger M, Aguzzi A. Research Institute of Molecular Pathology, Vienna, Austria. Medulloblatoma is a pediatric brain tumor originating in the human cerebellum. A collection of 23 medulloblastomas was analyzed for expression of the developmental control genes of the PAX and EN gene families by RNase protection and in situ hybridization. Of all nine PAX genes investigated, only PAX5 and PAX6 were consistently expressed in most medulloblastomas (70 and 78% of all cases, respectively), as were the genes EN1 (57%) and EN2 (78%). EN1, EN2, and PAX6 genes were also expressed in normal cerebellar tissue, and their expression in medulloblastoma is consistent with the hypothesis that this tumor originates in the external granular layer of the developing cerebellum. PAX5 transcripts were, however, not detected in the neonatal cerebellum, indicating that this gene is deregulated in medulloblastoma. In the desmoplastic variant of medulloblastoma, PAX5 expression was restricted to the reticulin-producing proliferating tumor areas containing undifferentiated cells; PAX5 was not expressed in the reticulin-free nonproliferating islands undergoing neuronal differentiation. These data suggest that deregulated expression of PAX5 correlates positively with cell proliferation and inversely with neuronal differentiation in desmoplastic medulloblastoma. PMID: 7777574 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: 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] ---------------------------------------------------------------