1: FASEB J. 2005 Sep;19(11):1537-9. Epub 2005 Jun 21. Serotonergic-like progenitor cells propagated from neural stem cells in vitro: survival with SERT protein expression following implantation into brains of mice lacking SERT. Ren-Patterson RF, Kim DK, Zheng X, Sherrill S, Huang SJ, Tolliver T, Murphy DL. Laboratory of Clinical Science, National Institute of Mental Health, NIH, Bethesda, MD 20892-1264, USA. renpatr@intra.nimh.nih.gov Neural stem cells (NSCs) obtained from the midbrain region of embryonic (E14) mice were initially cultured with basic fibroblast growth factor (bFGF), Sonic hedgehog, and FGF-8 in a serum-free N-2 culture medium to foster differentiation into a serotonergic-like phenotype. During the initial differentiating phase, these progenitor cells expressed En1, Pax3, and Pax5 mRNA. Subsequently, a single serotonin [5-hydroxytryptamine (5-HT)] and tryptophan hydroxylase-positive clone was isolated, which gave rise to cells that developed serotonergic properties. Sixty percent of these progenitor cells expressed the serotonin transporter (SERT), as indicated by specific ligand binding of [125I]-RTI-55. To further evaluate SERT functionality, we showed that these progenitor cells possessed specific [3H]-5-HT uptake activity. Implantation of the serotonergic-like progenitors into the hippocampus of adult mice genetically lacking SERT was followed by migration of these cells into adjacent brain regions, and survival of the cells at 8 weeks was accompanied by a gradual increase in density of SERT protein expression, which was not found in vehicle-injected, control mice. These findings suggest that this serotonergic-like NSC model will be a useful contribution to the development of cell biotechnology in regard to the expression of missing genes such as SERT in the adult brain. PMID: 15972295 [PubMed - in process] --------------------------------------------------------------- 2: Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12543-8. Epub 2004 Aug 13. Derivation of midbrain dopamine neurons from human embryonic stem cells. Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L. Laboratory of Stem Cell and Tumor Biology, Division of Neurosurgery and Developmental Biology Program, and Cell Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA. Human embryonic stem (hES) cells are defined by their extensive self-renewal capacity and their potential to differentiate into any cell type of the human body. The challenge in using hES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential toward the derivation of a specific cell fate. Within the nervous system, hES cells have been shown to differentiate in vitro into neural progenitor cells, neurons, and astrocytes. However, to our knowledge, the selective derivation of any given neuron subtype has not yet been demonstrated. Here, we describe conditions to direct hES cells into neurons of midbrain dopaminergic identity. Neuroectodermal differentiation was triggered on stromal feeder cells followed by regional specification by means of the sequential application of defined patterning molecules that direct in vivo midbrain development. Progression toward a midbrain dopamine (DA) neuron fate was monitored by the sequential expression of key transcription factors, including Pax2, Pax5, and engrailed-1 (En1), measurements of DA release, the presence of tetrodotoxin-sensitive action potentials, and the electron-microscopic visualization of tyrosinehydroxylase-positive synaptic terminals. High-yield DA neuron derivation was confirmed from three independent hES and two monkey embryonic stem cell lines. The availability of unlimited numbers of midbrain DA neurons is a first step toward exploring the potential of hES cells in preclinical models of Parkinson's disease. This experimental system also provides a powerful tool to probe the molecular mechanisms that control the development and function of human midbrain DA neurons. PMID: 15310843 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Nat Neurosci. 2001 Dec;4(12):1175-81. Distinct regulators control the expression of the mid-hindbrain organizer signal FGF8. Ye W, Bouchard M, Stone D, Liu X, Vella F, Lee J, Nakamura H, Ang SL, Busslinger M, Rosenthal A. Department of Molecular Biology, Genentech, 1 DNA Way, South San Francisco, California 94080, USA. Local expression of FGF8 at the mid/hindbrain boundary (MHB) governs the development of multiple neurons and support cells. Here we show that the paired-domain protein Pax2 is necessary and sufficient for the induction of FGF8 in part by regulating the expression of Pax5&8. A network of transcription and secreted factors, including En1, Otx2, Gbx2, Grg4 and Wnt1&4, that is established independently of Pax2, further refines the expression domain and level of FGF8 at the MHB through opposing effects on Pax2 activity. Our results indicate that the expression of local organizing factors is controlled by combinatorial interaction between inductive and modulatory factors. PMID: 11704761 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: 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] --------------------------------------------------------------- 5: 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] --------------------------------------------------------------- 6: 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] --------------------------------------------------------------- 7: 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] --------------------------------------------------------------- 8: Development. 1999 Nov;126(21):4827-38. FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate. Liu A, Losos K, 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. joyner@saturn.med.nyu.edu The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures (Crossley, P. H., Martinez, S. and Martin, G. R. (1996) Nature 380, 66-68). In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produced no apparent patterning defects in the embryos examined (Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997) Development 124, 959-969). We show here that FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. We also show that FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression. PMID: 10518499 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13870-5. The mouse Pax2(1Neu) mutation is identical to a human PAX2 mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney. Favor J, Sandulache R, Neuhauser-Klaus A, Pretsch W, Chatterjee B, Senft E, Wurst W, Blanquet V, Grimes P, Sporle R, Schughart K. GSF-National Research Center for Environment and Health, Institute of Mammalian Genetics, Oberschleissheim, Germany. We describe a new mouse frameshift mutation (Pax2(1Neu)) with a 1-bp insertion in the Pax2 gene. This mutation is identical to a previously described mutation in a human family with renal-coloboma syndrome [Sanyanusin, P., McNoe, L. A., Sullivan, M. J., Weaver, R. G. & Eccles, M. R. (1995) Hum. Mol. Genet. 4, 2183-2184]. Heterozygous mutant mice exhibit defects in the kidney, the optic nerve, and retinal layer of the eye, and in homozygous mutant embryos, development of the optic nerve, metanephric kidney, and ventral regions of the inner ear is severely affected. In addition, we observe a deletion of the cerebellum and the posterior mesencephalon in homozygous mutant embryos demonstrating that, in contrast to mutations in Pax5, which is also expressed early in the mid-hindbrain region, loss of Pax2 gene function alone results in the early loss of the mid-hindbrain region. The mid-hindbrain phenotype is similar to Wnt1 and En1 mutant phenotypes, suggesting the conservation of gene regulatory networks between vertebrates and Drosophila. PMID: 8943028 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: 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] ---------------------------------------------------------------