1: Brain Res Brain Res Rev. 2005 Sep;49(2):120-6. Epub 2005 Jan 21. Isthmus organizer for midbrain and hindbrain development. Nakamura H, Katahira T, Matsunaga E, Sato T. Department of Molecular Neurobiology, Graduate School of Life Sciences and Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan. nakamura@idac.tohoku.ac.jp Classical transplantation studies showed that the isthmus has an organizing activity upon the tectum and cerebellum. Since Fgf8 is expressed in the isthmus and mimics functionally isthmic grafts, it is accepted that Fgf8 plays pivotal role in the isthmic organizing activity. The fate of brain vesicles is determined by the combinations of transcription factors. The neural tube region where Otx2, Pax2, and En1 are expressed early on acquires midbrain identity. Pax3/7 forces the midbrain to differentiate into tectum. En1/2, Pax2/5, and Fgf8 form a positive feedback loop for their expression, thus misexpression of one of these molecules turns on the loop and forces presumptive diencephalon to differentiate into tectum. The isthmic organizer signal, Fgf8, stabilizes or changes the expression of the transcription factors in mid/hindbrain region. A strong Fgf8 signal activates the Ras-ERK signaling pathway, which in turn activates Irx2 in a rostrodorsal part of the hindbrain, and forces this tissue to differentiate into cerebellum. Publication Types: Review PMID: 16111543 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 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: J Neurochem. 2005 Mar;92(5):1265-76. In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons. Park CH, Minn YK, Lee JY, Choi DH, Chang MY, Shim JW, Ko JY, Koh HC, Kang MJ, Kang JS, Rhie DJ, Lee YS, Son H, Moon SY, Kim KS, Lee SH. Department of Microbiology, College of Medicine, Hanyang University, Seoul, Korea. Human embryonic stem (hES) cells, due to their capacity of multipotency and self-renewal, may serve as a valuable experimental tool for human developmental biology and may provide an unlimited cell source for cell replacement therapy. The purpose of this study was to assess the developmental potential of hES cells to replace the selectively lost midbrain dopamine (DA) neurons in Parkinson's disease. Here, we report the development of an in vitro differentiation protocol to derive an enriched population of midbrain DA neurons from hES cells. Neural induction of hES cells co-cultured with stromal cells, followed by expansion of the resulting neural precursor cells, efficiently generated DA neurons with concomitant expression of transcriptional factors related to midbrain DA development, such as Pax2, En1 (Engrailed-1), Nurr1, and Lmx1b. Using our procedure, the majority of differentiated hES cells (> 95%) contained neuronal or neural precursor markers and a high percentage (> 40%) of TuJ1+ neurons was tyrosine hydroxylase (TH)+, while none of them expressed the undifferentiated ES cell marker, Oct 3/4. Furthermore, hES cell-derived DA neurons demonstrated functionality in vitro, releasing DA in response to KCl-induced depolarization and reuptake of DA. Finally, transplantation of hES-derived DA neurons into the striatum of hemi-parkinsonian rats failed to result in improvement of their behavioral deficits as determined by amphetamine-induced rotation and step-adjustment. Immunohistochemical analyses of grafted brains revealed that abundant hES-derived cells (human nuclei+ cells) survived in the grafts, but none of them were TH+. Therefore, unlike those from mouse ES cells, hES cell-derived DA neurons either do not survive or their DA phenotype is unstable when grafted into rodent brains. PMID: 15715675 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: 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] --------------------------------------------------------------- 5: Cerebellum. 2004;3(1):16-20. Retinoids and binding proteins in the cerebellum during lifetime. Parenti R, Cicirata F. Department of Physiological Sciences, University of Catania, Catania, Italy. parenti@mbox.unict.it The vertebrate nervous system is one of the main sites where retinoids and related binding proteins play their critical roles. The cerebellum is subjected to the effects of retinoids over its lifetime. The retinoid binding proteins are differently expressed during life: CRBP I, expressed in the adult cerebellum, is possibly involved in plasticity during memory processes. CRABP I, expressed in the embryonic cerebellum, is involved in the development of the organ and in cellular differentiation. In particular, since the expression of CRABP I coincides with earliest cerebellar differentiation, it is probably involved in this critical step, participating in a complex network of regulatory genes expressed in the mes/methencephalon such as Fgf8, Wnt1, En1/2, Pax2/5, Otx2 and Gbx2. Finally, the way for metabolic synchronization of isthmic cell populations and the putative existence of alternative pathways for CRABP I to regulate RA are discussed. Publication Types: Review Review, Tutorial PMID: 15072263 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Neurobiol. 2004 Apr;59(1):48-56. Polarity and laminar formation of the optic tectum in relation to retinal projection. Nakamura H, Sugiyama S. Department of Molecular Neurobiology, Graduate School of Life Sciences and Institute of Development Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan. nakamura@idac.tohoku.ac.jp The mes-metencephalic boundary (isthmus) works as an organizer for the tectum, and the organizing molecule may be Fgf8. The region where Otx2, En1, and Pax2 are expressed overlappingly may differentiate into the mesencephalon. The di-mesencephalic and mes-metencephalic boundaries are determined by repressive interaction of Pax6 and En1/Pax2 and of Otx2 and Gbx2, respectively. The optic tectum is a visual center in lower vertebrates. The tectum and the retina should be regionalized and be positionally specialized for the proper retinotopic projection. Gradient of En2 plays a crucial role in rostrocaudal polarity formation of the tectum. En2 confers caudal characteristics of the retina by inducing ephrinA2 and A5, which are the repellant molecules for the growth cones of temporal retinal ganglion cells. Grg4 antagonizes the isthmus-related genes, and is involved in the formation of di-mesencephalic boundary and tectal polarity formation at an early phase of development. Then, Grg4 plays a role in tectal laminar formation by controlling the migration pathway. Migration pathway of tectal postmitotic cells changes after E5. The late migratory cells split the early migratory neurons to form laminae h-j of SGFS. Grg4 is expressed in the ventricular layer after E5, and forces postmitotic cells to follow the late migratory pathway, though retinal fibers terminate at laminae a-f of SGFS. Misexpression of Grg4 disrupts the lamina g, and in such tecta retinal arbors invade deep into the tectal layer, indicating that lamina g is a nonpermissive lamina for the retinal arbors. Copyright 2004 Wiley Periodicals, Inc. J Neurobiol 59: 48-56, 2004 Publication Types: Review Review, Tutorial PMID: 15007826 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Development. 2001 Dec;128(23):4789-800. Regionalisation of anterior neuroectoderm and its competence in responding to forebrain and midbrain inducing activities depend on mutual antagonism between OTX2 and GBX2. Martinez-Barbera JP, Signore M, Boyl PP, Puelles E, Acampora D, Gogoi R, Schubert F, Lumsden A, Simeone A. MRC Centre for Developmental Neurobiology, King's College London, Guy's Campus, New Hunt's House, London SE1 1UL, UK. The anterior neural ridge (ANR), and the isthmic organiser (IsO) represent two signalling centres possessing organising properties necessary for forebrain (ANR) as well as midbrain and rostral hindbrain (IsO) development. An important mediator of ANR and IsO organising property is the signalling molecule FGF8. Previous work has indicated that correct positioning of the IsO and Fgf8 expression in this domain is controlled by the transcription factors Otx2 and Gbx2. In order to provide novel insights into the roles of Otx2 and Gbx2, we have studied mutant embryos carrying different dosages of Otx2, Otx1 and Gbx2. Embryos deficient for both OTX2 and GBX2 proteins (hOtx1(2)/hOtx1(2); Gbx2(-/-)) show abnormal patterning of the anterior neural tissue, which is evident at the presomite-early somite stage prior to the onset of Fgf8 neuroectodermal expression. Indeed, hOtx1(2)/hOtx1(2); Gbx2(-/-) embryos exhibit broad co-expression of early forebrain, midbrain and rostral hindbrain markers such as hOtx1, Gbx2, Pax2, En1 and Wnt1 and subsequently fail to activate forebrain and midbrain-specific gene expression. In this genetic context, Fgf8 is expressed throughout the entire anterior neural plate, thus indicating that its activation is independent of both OTX2 and GBX2 function. Analysis of hOtx1(2)/hOtx1(2); Gbx2(-/-) and Otx1(+/-); Otx2(+/-) mutant embryos also suggests that FGF8 cannot repress Otx2 without the participation of GBX2. Finally, we report that embryos carrying a single strong hypomorphic Otx2 allele (Otx2(lambda)) in an Otx2 and Gbx2 null background (Otx2(lambda)/-; Gbx2(-/-)) recover both the headless phenotype exhibited by Otx2(lambda)/- embryos and forebrain- and midbrain-specific gene expression that is not observed in hOtx1(2)/hOtx1(2); Gbx2(-/-) mutants. Together, these data provide novel genetic evidence indicating that OTX2 and GBX2 are required for proper segregation of early regional identities anterior and posterior to the mid-hindbrain boundary (MHB) and for conferring competence to the anterior neuroectoderm in responding to forebrain-, midbrain- and rostral hindbrain-inducing activities. PMID: 11731459 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: 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] --------------------------------------------------------------- 9: 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] --------------------------------------------------------------- 10: Int J Dev Biol. 2001;45(1):367-71. The isthmic organizer and brain regionalization. Martinez S. Instituto de Neurociencias, Universidad Miguel Hernandez, Alicante, Spain. smartinez@mussol.umh.es Distinct neural identities are acquired through progressive restriction of developmental potential under the influence of local environmental signals. Evidence for the localization of such morphogenetic signals at specific locations of the developing neural primordium has suggested the concept of "secondary organizer regions", which regulate the identity and regional polarity of neighboring neuroepithelial areas one step further. In recent years, the most studied secondary organizer has been the isthmic organizer, which is localized at the hind-midbrain transition and controls anterior hindbrain and midbrain regionalization. Otx2 and Gbx2 expression is fundamental for positioning the organizer and for the establishment of molecular interactions that induce Fgf8 expression and then, stabilize the autoregulative loop of En1, Wnt1 and Pax2 expression. Temporospatial patterns of such gene expressions are necessary for the correct development of the organizer which, by a planar mechanism of induction, controls the normal development of the rostral hindbrain from r2 to the midbrain-diencephalic boundary. Fgf8 appears as the active diffusible molecule for isthmic morphogenetic activity and has been suggested to be the morphogenetic effector in other inductive activities revealed in other neuroepithelial regions. Publication Types: Review Review, Tutorial PMID: 11291867 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Mech Dev. 2001 Mar;101(1-2):111-8. Neuroepithelial co-expression of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer. Garda AL, Echevarria D, Martinez S. Instituto de Neurociencias, Fac. de Medicina, Campus de San Juan (UMH), E-03550 -, Alicante, Spain. The most studied secondary neural organizer is the isthmic organizer, which is localized at the mid-hindbrain transition of the neural tube and controls the anterior hindbrain and midbrain regionalization. Otx2 and Gbx2 expressions are fundamental for positioning the organizer and the establishment of molecular interactions that induce Fgf8. We present here evidences demonstrating that Otx2 and Gbx2 have an overlapping expression in the isthmic region. This area is the transversal domain where expression of Fgf8 is induced. The Fgf8 protein produced in the isthmus stabilizes and up-regulates Gbx2 expression, which, in turn, down-regulates Otx2 expression. The inductive effect of the Gbx2/Otx2 limit keeps Fgf8 expression stable and thus maintains its positive role in the expression of Pax2, En1,2 and Wnt1. PMID: 11231064 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: 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] --------------------------------------------------------------- 13: Biochem Cell Biol. 2000;78(5):577-84. Regulation and function of FGF8 in patterning of midbrain and anterior hindbrain. Mason I, Chambers D, Shamim H, Walshe J, Irving C. MRC Centre for Developmental Neurobiology, King's College London, UK. ivor.mason@kcl.ac.uk In this article, an adjunct to a platform presentation at the Winternational 2000 Symposium, we summarize the recent findings of this group concerning the regulation and functions of FGF8 expressed at the isthmus of the developing brain. We show that several different FGF8 isoforms, ectopically expressed in midbrain or posterior forebrain, are able to mimic the proliferative and patterning functions previously attributed to the isthmus in tissue grafting studies. Moreover, we also show that FGF8 protein is sufficient to induce an ectopic isthmic organiser (Fgf-8+, Gbx2+) in anterior midbrain. We also provide evidence that isthmic FGF8 patterns anterior hindbrain, repressing Hox-a2 expression and setting aside a territory of the brain that includes the cerebellar anlage. We show that these effects of FGF8 are likely to be mediated via FGFR1 and be modulated by the putative FGF antagonist, Sprouty2, identified using a differential display screen. Finally, we provide evidence that the onset of Fgf8 expression is regulated by En1 and that its expression at the isthmus is subsequently maintained by a specific and direct interaction between rhombomere 1 and midbrain. PMID: 11103948 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Dev Neurosci. 2000;22(3):207-16. Sonic hedgehog-mediated ventralization disrupts formation of the midbrain-hindbrain junction in the chick embryo. Zhang XM, Lin E, Yang XJ. Department of Neurobiology, Department of Ophthalmology and Jules Stein Eye Institute, University of California at Los Angeles, 90095, USA. yang@jsei.ucla.edu The secreted signaling molecule Sonic hedgehog (Shh) plays critical roles in pattern formation of the vertebrate central nervous system. During neurulation, Shh is produced by the ventral midline mesoderm as well as by the ventral neural tube, and its activity is required for the determination of ventral characteristics along the anterior-posterior neuraxis. The morphological boundary between midbrain and hindbrain, the isthmus, is an important tissue organizer that regulates the development of both the midbrain and the anterior hindbrain. In this study, we report that retrovirus-mediated misexpression of Shh in the early chick neural tube disrupts formation of the boundary between the midbrain and the hindbrain, and causes a fusion of the bilateral cerebellum primordia. Dorsally expressed Shh signals induce ectopic transcription of its receptor Ptc1 in the midbrain and the hindbrain. The expression of several genes (Noggin, Lmx1, BMP7) along the dorsal midline of the midbrain is abolished, and ventral or lateral markers (HNF3 beta, Ptc1, ELF1) are induced in the dorsal brain. Furthermore, the normally restricted expression of two genes (En1 and Pax2) in the mid/hindbrain junction region are expanded, reflecting the morphological defects. These results suggest that maintaining proper dorsal-ventral patterns of the neural tube is essential for normal development of the mid/hindbrain region. Copyright 2000 S. Karger AG, Basel PMID: 10894984 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: 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] --------------------------------------------------------------- 16: Nature. 1999 Sep 9;401(6749):164-8. The caudal limit of Otx2 expression positions the isthmic organizer. Broccoli V, Boncinelli E, Wurst W. GSF-Research Centre for Environment and Health, Institute of Mammalian Genetics, Neuherberg, Germany. The homeobox gene Otx2 is expressed in the anterior neural tube with a sharp limit at the midbrain/hindbrain junction (the isthmic organizer). Otx2 inactivation experiments have shown that this gene is essential for the development of its expression domain. Here we investigate whether the caudal limit of Otx2 expression is instrumental in positioning the isthmic organizer and in specifying midbrain versus hindbrain fate, by ectopically expressing Otx2 in the presumptive anterior hindbrain using a knock-in strategy into the En1 locus. Transgenic offspring display a cerebellar ataxia. Morphological and histological studies of adult transgenic brains reveal that most of the anterior cerebellar vermis is missing, whereas the inferior colliculus is complementarily enlarged. During early neural pattern formation expression of the midbrain markers Wnt1 and Ephrin-A5, the isthmic organizer markers Pax2 and Fgf-8 and the hindbrain marker Gbx2 are shifted caudally in the presumptive hindbrain territory. These findings show that the caudal limit of Otx2 expression is sufficient for positioning the isthmic organizer and encoding caudal midbrain fate within the mid/hindbrain domain. PMID: 10490025 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Development. 1999 Feb;126(5):945-59. Sequential roles for Fgf4, En1 and Fgf8 in specification and regionalisation of the midbrain. Shamim H, Mahmood R, Logan C, Doherty P, Lumsden A, Mason I. MRC Brain Development Programme, Department of Developmental Neurobiology, Medical School's of Guy's, King's and St. Thomas's Hospitals, King's College London, London SE1 9RT, UK. Experiments involving tissue recombinations have implicated both early vertical and later planar signals in the specification and polarisation of the midbrain. Here we investigate the role of fibroblast growth factors in regulating these processes in the avian embryo. We show that Fgf4 is expressed in the notochord anterior to Hensen's node before transcripts for the earliest molecular marker of midbrain tissue in the avian embryo, En1, are detected. The presence of notochord is required for the expression of En1 in neural plate explants in vitro and FGF4 mimics this effect of notochord tissue. Subsequently, a second member of the fibroblast growth factor family, Fgf8, is expressed in the isthmus in a manner consistent with it providing a polarising signal for the developing midbrain. Using a retroviral vector to express En1 ectopically, we show that En1 can induce Fgf8 expression in midbrain and posterior diencephalon. Results of the introduction of FGF8 protein into the anterior midbrain or posterior diencephalon are consistent with it being at least part of the isthmic activity which can repolarise the former tissue and respecify the latter to a midbrain fate. However, the ability of FGF8 to induce expression of genes which have earlier onsets of expression than Fgf8 itself, namely En1 and Pax2, strongly suggests that the normal function of FGF8 is in maintaining patterns of gene expression in posterior midbrain. Finally, we provide evidence that FGF8 also provides mitogenic stimulation during avian midbrain development. PMID: 9927596 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Development. 1997 Nov;124(22):4493-503. PAX2 is expressed in multiple spinal cord interneurons, including a population of EN1+ interneurons that require PAX6 for their development. Burrill JD, Moran L, Goulding MD, Saueressig H. Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, CA 92037, USA. Members of the PAX family of transcription factors are candidates for controlling cell identity in the spinal cord. We have morphologically analyzed cells that express one of these transcription factors, PAX2, demonstrating multiple interneuron cell types express PAX2. Two ventral populations of PAX2-expressing interneurons in the spinal cord are marked by coexpression of the transcription factors, EN1 and EVX1. Interestingly, the expression domains of PAX2, EN1 and EVX1 in postmitotic neurons correlate closely with those of Pax6 and Pax7 in the ventricular zone, implicating these patterning genes in the regulation of PAX2, EN1 and EVX1. We show that one of these patterning genes, Pax6, is required for the correct specification of ventral PAX2+ interneurons that coexpress EN1. These results demonstrate that the early activity of patterning genes in the ventricular zone determines interneuron identity in the spinal cord. PMID: 9409667 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: 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] ---------------------------------------------------------------