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Harukazu Nakamura - One of the best experts on this subject based on the ideXlab platform.

  • Activation of Ras-ERK pathway by Fgf8 and its downregulation by Sprouty2 for the isthmus organizing activity.
    Developmental Biology, 2009
    Co-Authors: Asuka Suzuki-hirano, Hidekiyo Harada, Tatsuya Sato, Harukazu Nakamura
    Abstract:

    Abstract In the previous studies, we showed that strong Fgf8 signaling activates the Ras-ERK pathway to induce cerebellum. Here, we show importance of negative regulation following activation of this pathway for proper regionalization of mesencephalon and metencephalon in chick embryos. ‘Prolonged’ activation of ERK by Misexpression of Fgf8b and dominant-negative Sprouty2 (dnSprouty2) did not change the fate of the mesencephalic alar plate. Downregulation of ERK activity using an MEK inhibitor, U0126, or by tetracycline-dependent Tet-off system after co-expression of Fgf8b and dnSprouty2 forced the mesencephalic alar plate to differentiate into cerebellum. We then paid attention to Mkp3. After Misexpression of dnMkp3 and Fgf8b, slight downregulation of ERK activity occurred, which may be due to Sprouty2, and the mesencephalon transformed to the isthmus-like structure. The results indicate that ERK must be once upregulated and then be downregulated for cerebellar differentiation and that differential ERK activity level established by negative regulators receiving Fgf8 signal may determine regional specificity of mesencephalon and metencephalon.

  • Activation of Ras-ERK pathway by Fgf8 and its downregulation by Sprouty2 for the isthmus organizing activity
    Nature Precedings, 2008
    Co-Authors: Asuka Suzuki-hirano, Tatsuya Sato, Harukazu Nakamura
    Abstract:

    In the previous studies, we showed that strong Fgf8 signaling activates the Ras-ERK pathway to induce cerebellum. Here, we show importance of negative regulation of this pathway. 'Prolonged' activation of ERK by Misexpression of Fgf8b and dominant-negative Sprouty2 ( dnSprouty2 ) did not change the fate of the mesencephalic alar plate. Downregulation of ERK activity using a MEK inhibitor, U0126, or by tetracycline dependent Tet-off system after co-expression of Fgf8b and dnSprouty2 , forced the mesencephalic alar plate to differentiate into cerebellum. We then paid attention to Mkp3. After Misexpression of dnMkp3 and Fgf8b , slight downregulation of ERK activity occurred, which may be due to Sprouty2, and the mesencephalon transformed to the isthmus-like structure. The results indicate that ERK must be once upregulated and then be downregulated for cerebellar differentiation, and that differential ERK activity level established by negative regulators receiving Fgf8 signal may determine regional specificity of mesencephalon and metencephalon.

  • Regulation of isthmic Fgf8 signal by sprouty2.
    Development (Cambridge England), 2004
    Co-Authors: Asuka Suzuki-hirano, Tatsuya Sato, Harukazu Nakamura
    Abstract:

    Fgf8 functions as an organizer at the mes/metencephalic boundary (isthmus). We showed that a strong Fgf8 signal activates the Ras-ERK signaling pathway to organize cerebellar differentiation. Sprouty2 is expressed in an overlapping manner to Fgf8, and is induced by Fgf8. Its function, however, is indicated to antagonize Ras-ERK signaling. Here, we show the regulation of Fgf8 signaling in relation to Sprouty2. sprouty2 expression was induced very rapidly by Fgf8b, but interfered with ERK activation. sprouty2 Misexpression resulted in a fate change of the presumptive metencephalon to the mesencephalon. Misexpression of a dominant negative form of Sprouty2 augmented ERK activation, and resulted in anterior shift of the posterior border of the tectum. The results indicate that Fgf8 activates the Ras-ERK signaling pathway to differentiate the cerebellum, and that the hyper- or hypo-signaling of this pathway affects the fate of the brain vesicles. Sprouty2 may regulate the Fgf8-Ras-ERK signaling pathway for the proper regionalization of the metencephalon and mesencephalon.

  • Role of Lmx1b and Wnt1 in mesencephalon and metencephalon development.
    Development (Cambridge England), 2002
    Co-Authors: Eiji Matsunaga, Tatsuya Katahira, Harukazu Nakamura
    Abstract:

    The isthmus is the organizing center for the tectum and cerebellum. Fgf8 and Wnt1 are secreted molecules expressed around the isthmus. The function of Fgf8 has been well analyzed, and now accepted as the most important organizing signal. Involvement of Wnt1 in the isthmic organizing activity was suggested by analysis of Wnt1 knockout mice. But its role in isthmic organizing activity is still obscure. Recently, it has been shown that Lmx1b is expressed in the isthmic region and that it may occupy higher hierarchical position in the gene expression cascade in the isthmus. We have carried out Misexpression experiment of Lmx1b and Wnt1, and considered their role in the isthmic organizing activity. Lmx1b or Wnt1 Misexpression caused expansion of the tectum and cerebellum. Fgf8 was repressed in a cells that misexpress Lmx1b, but Fgf8 expression was induced around Lmx1b-misexpressing cells. As Lmx1b induced Wnt1 and Wnt1 induced Fgf8 expression in turn, Wnt1 may be involved in non cell-autonomous induction of Fgf8 expression by Lmx1b. Wnt1 could not induce Lmx1b expression so that Lmx1b may be put at the higher hierarchical position than Wnt1 in gene expression cascade in the isthmus. We have examined the relationship among isthmus related genes, and discuss the mechanism of the formation and maintenance of isthmic organizing activity.

  • Regionalisation and acquisition of polarity in the optic tectum.
    Progress in Neurobiology, 2001
    Co-Authors: Harukazu Nakamura
    Abstract:

    Abstract 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.

Clifford J. Tabin - One of the best experts on this subject based on the ideXlab platform.

  • Genomes & Developmental Control Sox9 and Nkx2.5 determine the pyloric sphincter epithelium under the control of BMP signaling
    2005
    Co-Authors: Nicole A. Theodosiou, Clifford J. Tabin
    Abstract:

    The organs of the digestive tract are specified by coordinated signaling between the endoderm and mesoderm during development. These epithelial–mesenchymal interactions lead to the organ-specific morphogenesis and differentiation of regions along the gut tube. In this paper, we show that in the chick, the SRY-related transcription factor Sox9 is a marker for the posterior gizzard. Viral Misexpression of Sox9 in the gizzard mesoderm is sufficient to specify epithelium characteristic of the pyloric sphincter. Sox9 expression is normally limited to the region of the posterior gizzard under the regulation of BMP signaling from the adjacent midgut. Misexpression of an activated form of BMPR1b in the gizzard upregulates Sox9 expression, while the BMP antagonist noggin down-regulates Sox9 expression in the gizzard mesoderm. Previously, Nkx2.5 was identified as a marker for the mesoderm of the pyloric sphincter. As with Sox9, BMP signaling appears to regulate Nkx2.5 and its ability to determine the pyloric epithelium. Despite these similarities, our evidence suggests that Sox9 and Nkx2.5 are regulated independently by BMP signaling, and act coordinately to specify the pyloric sphincter.

  • Pax3 and Dach2 positive regulation in the developing somite.
    Developmental dynamics : an official publication of the American Association of Anatomists, 2002
    Co-Authors: Gabrielle Kardon, Tiffany A. Heanue, Clifford J. Tabin
    Abstract:

    In vertebrates, skeletal muscles of the body arise from cells of somitic origin. Recently, somite culture experiments have identified a set of genes, including Pax3, Six1, Eya2, and Dach2, that appear to play an important role in early myogenesis during somite development (Heanue et al. [1999] Genes Dev. 13:3231-3243). In somite culture Pax3, Six1, Eya2, and Dach2 not only function to activate myogenesis, but they form a complex network regulating each other's transcription. We sought to examine whether this putative Pax3/Six1/Eya2/Dach2 network of regulation actually functions in vivo. In particular, we tested whether Pax3 and Dach2 participate in a positive regulatory feedback loop in vivo as they do in culture. To test in vivo Pax3/Dach2 interregulation, we took advantage of the known dependence of both factors on ectodermal signals. Somites isolated from the overlying ectoderm lose expression of Pax3 and Dach2. Therefore, we attempted to rescue Pax3 or Dach2 expression in somites isolated from the ectoderm by retroviral Misexpression of the complementary factor. Indeed Misexpression of Pax3 or Dach2 resulted in rescue of Dach2 or Pax3, respectively. These rescue experiments demonstrate that Pax3 and Dach2 positively regulate each other's expression in vivo and support the validity of the Pax3/Six1/Eya2/Dach 2 network in regulating myogenesis.

  • dual roles of wnt signaling during chondrogenesis in the chicken limb
    Development, 2000
    Co-Authors: Christine Hartmann, Clifford J. Tabin
    Abstract:

    Long bones of the appendicular skeleton are formed from a cartilage template in a process known as endochondral bone development. Chondrocytes within this template undergo a progressive program of differentiation from proliferating to postmitotic prehypertrophic to hypertrophic chondrocytes, while mesenchymal cells immediately surrounding the early cartilage template form the perichondrium. Recently, members of the Wnt family of secreted signaling molecules have been implicated in regulating chondrocyte differentiation. We find that Wnt-5a, Wnt-5b and Wnt-4 genes are expressed in chondrogenic regions of the chicken limb: Wnt-5a is expressed in the perichondrium, Wnt-5b is expressed in a subpopulation of prehypertrophic chondrocytes and in the outermost cell layer of the perichondrium, and Wnt-4 is expressed in cells of the joint region. Misexpression experiments demonstrate that two of these Wnt molecules, Wnt-5a and Wnt-4, have opposing effects on the differentiation of chondrocytes and that these effects are mediated through divergent signaling pathways. Specifically, Wnt-5a Misexpression delays the maturation of chondrocytes and the onset of bone collar formation, while Wnt-4 Misexpression accelerates these two processes. Misexpression of a stabilized form of beta-catenin also results in accelerated chondrogenesis, suggesting that a beta-catenin/TCF-LEF complex is involved in mediating the positive regulatory effect of Wnt-4. A number of the genes involved in Wnt signal tranduction, including two members of the Frizzled gene family, which are believed to encode Wnt-receptors, show very dynamic and distinct expression patterns in cartilaginous elements of developing chicken limbs. Misexpression of putative dominant-negative forms of the two Frizzled proteins results in severe shortening of the infected cartilage elements due to a delay in chondrocyte maturation, indicating that an endogenous Wnt signal does indeed function to promote chondrogenic differentiation.

  • Targeted Gene Misexpression in Chick Limb Buds Using Avian Replication-Competent Retroviruses
    Methods (San Diego Calif.), 1998
    Co-Authors: Malcolm Logan, Clifford J. Tabin
    Abstract:

    The methods and applications for using avian replication-competent retroviruses to target gene Misexpression in the developing limb bud of the chicken are described. These viruses provide the means to exploit the strengths of the chick as a model system in experimental embryology in conjunction with a genetic approach for ectopically expressing a gene of interest. The applications and strengths of the system are detailed. All the steps required to produce a virus carrying a transgene of interest and the methodologies behind designing and carrying out Misexpression strategies are outlined, and some useful techniques for analyzing infected embryos are described.

  • Analysis of Hoxd-13 and Hoxd-11 Misexpression in chick limb buds reveals that Hox genes affect both bone condensation and growth
    Development (Cambridge England), 1997
    Co-Authors: Deborah J. Goff, Clifford J. Tabin
    Abstract:

    Hox genes are important regulators of limb pattern in vertebrate development. Misexpression of Hox genes in chicks using retroviral vectors provides an opportunity to analyze gain-of-function phenotypes and to assess their modes of action. Here we report the Misexpression phenotype for Hoxd-13 and compare it to the Misexpression phenotype of Hoxd-11. Hoxd-13 Misexpression in the hindlimb results in a shortening of the long bones, including the femur, the tibia, the fibula and the tarsometatarsals. Mutations in an alanine repeat region in the N-terminus of Hoxd-13 have recently been implicated in human synpolydactyly (Muragaki, Y., Mundlos, S., Upton, J. and Olsen, B. R. (1996) Science 272, 548–551). N-terminal truncations of Hoxd-13 which lack this repeat were constructed and were found to produce a similar, although slightly milder, Misexpression phenotype than the full-length Hoxd-13. The stage of bone development regulated by Hox genes has not previously been examined. The changes in bone lengths caused by Hoxd-13 Misexpression are late phenotypes that first become apparent during the growth phase of the bones. Analysis of tritiated thymidine uptake by the tibia and fibula demonstrates that Hox genes can pattern the limb skeleton by regulating the rates of cell division in the proliferative zone of growing cartilage. Hoxd-11, in contrast to Hoxd-13, acts both at the initial cartilage condensation phase in the foot and during the later growth phase in the lower leg. Ectopic Hoxd-13 appears to act in a dominant negative manner in regions where it is not normally expressed. We propose a model in which all Hox genes are growth promoters, regulating the expression of the same target genes, with some Hox genes being more effective promoters of growth than other Hox genes. According to this model, the overall rate of growth in a given region is the result of the combined action of all of the Hox genes expressed in that region competing for the same target genes.

David R. Soll - One of the best experts on this subject based on the ideXlab platform.

  • Misexpression of the opaque-phase-specific gene PEP1 (SAP1) in the white phase of Candida albicans confers increased virulence in a mouse model of cutaneous infection.
    Infection and immunity, 1999
    Co-Authors: Christopher Kvaal, Salil A. Lachke, Thyagarajan Srikantha, Karla J. Daniels, James J. Mccoy, David R. Soll
    Abstract:

    Candida albicans WO-1 switches reversibly and at high frequency between a white and an opaque colonyforming phenotype that includes dramatic changes in cell morphology and physiology. A Misexpression strategy has been used to investigate the role of the opaque-phase-specific gene PEP1 (SAP1), which encodes a secreted aspartyl proteinase, in the expression of the unique opaque-phase phenotype and phase-specific virulence in two animal models. The PEP1 (SAP1) open reading frame was inserted downstream of the promoter of the white-phase-specific gene WH11 in the transforming vector pCPW7, and the resulting transformants were demonstrated to misexpress PEP1 (SAP1) in the white phase. Misexpression did not confer any of the unique morphological characteristics of the opaque phase to cells in the white phase and had no effect on the switching process. However, Misexpression conferred upon white-phase cells the increased capacity of opaque-phase cells to grow in medium in which protein was the sole nitrogen source. Misexpression of PEP1 (SAP1) had no effect on the virulence of white-phase cells in a systemic mouse model, in which white-phase cells were already more virulent than opaque-phase cells. Misexpression did, however, confer upon white-phase cells the dramatic increase in colonization of skin in a cutaneous mouse model that was exhibited by opaque-phase cells. Misexpression of PEP1 (SAP1) conferred upon white-phase cells two dissociable opaque-phase characteristics: increased adhesion and the capacity to cavitate skin. The addition of pepstatin A to the cutaneous model inhibited the latter, but not the former, suggesting that the latter is effected by released enzyme, while the former is effected by cell-associated enzyme.

  • Misexpression of the white-phase-specific gene WH11 in the opaque phase of Candida albicans affects switching and virulence
    Infection and immunity, 1997
    Co-Authors: Christopher Kvaal, Thyagarajan Srikantha, David R. Soll
    Abstract:

    Candida albicans WO-1 switches between a white- and an opaque-colony-forming phenotype. The gene WH11 is expressed differentially in the white phase. The WH11 open reading frame was inserted downstream of the promoter of the opaque-phase-specific gene OP4 in the transforming vector pCWOP16, and resulting transformants were demonstrated to misexpress WH11 in the opaque phase. Misexpression had no effect on the ability to switch from the white to the opaque or the opaque to the white phase, and it had no effect on the genesis of the unique opaque-phase cellular phenotype, even though the Wh11 protein was distributed throughout the cytoplasm in a manner similar to that observed for the endogenous gene product in the white phase. Misexpression did, however, increase the frequency of the opaque-to-white transition 330-fold and markedly increased the virulence of cells in the opaque phase in a mouse tail injection model.

John V Brigande - One of the best experts on this subject based on the ideXlab platform.

  • functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer
    Nature, 2008
    Co-Authors: Samuel P Gubbels, David W Woessner, John C Mitchell, Anthony J Ricci, John V Brigande
    Abstract:

    Cochlear hair cells form the sound-sensing apparatus of vertebrates and their loss or damage results in hearing impairment. Mammals cannot regenerate these cells, but previous work has shown that ectopic expression of the transcription factor Atonal homologue 1 (Atoh1) can induce cells that would not normally differentiate as cochlear hair cells to become hair cell-like. Now Gubbels et al. show that in utero gene transfer of Atoh1 into mouse cochleas generates ectopic hair cells in the cochlea. Importantly, these supernumerary hair cells are functionally competent and display neuronal connectivity. This is a major step towards experiments to test for the ability of gene therapies to ameliorate hearing loss in mouse models of human deafness. Sensory hair cells in the mammalian cochlea convert mechanical stimuli into electrical impulses that subserve audition1,2. Loss of hair cells and their innervating neurons is the most frequent cause of hearing impairment3. Atonal homologue 1 (encoded by Atoh1, also known as Math1) is a basic helix–loop–helix transcription factor required for hair-cell development4,5,6, and its Misexpression in vitro7,8 and in vivo9,10 generates hair-cell-like cells. Atoh1-based gene therapy to ameliorate auditory10 and vestibular11 dysfunction has been proposed. However, the biophysical properties of putative hair cells induced by Atoh1 Misexpression have not been characterized. Here we show that in utero gene transfer of Atoh1 produces functional supernumerary hair cells in the mouse cochlea. The induced hair cells display stereociliary bundles, attract neuronal processes and express the ribbon synapse marker carboxy-terminal binding protein 2 (refs 12,13). Moreover, the hair cells are capable of mechanoelectrical transduction1,2 and show basolateral conductances with age-appropriate specializations. Our results demonstrate that manipulation of cell fate by transcription factor Misexpression produces functional sensory cells in the postnatal mammalian cochlea. We expect that our in utero gene transfer paradigm will enable the design and validation of gene therapies to ameliorate hearing loss in mouse models of human deafness14,15.

  • functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer
    Nature, 2008
    Co-Authors: Samuel P Gubbels, David W Woessner, John C Mitchell, Anthony J Ricci, John V Brigande
    Abstract:

    Sensory hair cells in the mammalian cochlea convert mechanical stimuli into electrical impulses that subserve audition. Loss of hair cells and their innervating neurons is the most frequent cause of hearing impairment. Atonal homologue 1 (encoded by Atoh1, also known as Math1) is a basic helix-loop-helix transcription factor required for hair-cell development, and its Misexpression in vitro and in vivo generates hair-cell-like cells. Atoh1-based gene therapy to ameliorate auditory and vestibular dysfunction has been proposed. However, the biophysical properties of putative hair cells induced by Atoh1 Misexpression have not been characterized. Here we show that in utero gene transfer of Atoh1 produces functional supernumerary hair cells in the mouse cochlea. The induced hair cells display stereociliary bundles, attract neuronal processes and express the ribbon synapse marker carboxy-terminal binding protein 2 (refs 12,13). Moreover, the hair cells are capable of mechanoelectrical transduction and show basolateral conductances with age-appropriate specializations. Our results demonstrate that manipulation of cell fate by transcription factor Misexpression produces functional sensory cells in the postnatal mammalian cochlea. We expect that our in utero gene transfer paradigm will enable the design and validation of gene therapies to ameliorate hearing loss in mouse models of human deafness.

Gregor Bucher - One of the best experts on this subject based on the ideXlab platform.

  • Heat shock-mediated Misexpression of genes in the beetle Tribolium castaneum
    Development Genes and Evolution, 2012
    Co-Authors: Johannes Benno Schinko, Kathrin Hillebrand, Gregor Bucher
    Abstract:

    Insect gene function has mainly been studied in the fruit fly Drosophila melanogaster because in this species many techniques and resources are available for gene knock down and the ectopic activation of gene function. However, in order to study biological aspects that are not represented by the Drosophila model, and in order to test to what degree gene functions are conserved within insects and what changes in gene function accompanied the evolution of novel traits, the establishment of respective tools in other insect species is required. While gene knock down can be induced by RNA interference in many insects, methods to misexpress genes are much less developed. In order to allow Misexpression of genes in a timely controlled manner in the red flour beetle Tribolium castaneum , we have established a heat shock-mediated Misexpression system. We show that endogenous heat shock elements perform better than artificial heat shock elements derived from vertebrates. We carefully determine the optimal conditions for heat shock and define a core promoter for use in future constructs. Finally, using this system, we study the effects of misexpressing the head patterning gene Tc-orthodenticle1 ( Tc-otd1 ), We show that Tc-otd1 suppresses Tc-wingless ( Tc-wg ) in the trunk and to some degree in the head.

  • functionality of the gal4 uas system in tribolium requires the use of endogenous core promoters
    BMC Developmental Biology, 2010
    Co-Authors: Johannes B Schinko, Markus Weber, Ivana Viktorinova, Alexandros Kiupakis, Michalis Averof, Martin Klingler, Ernst A Wimmer, Gregor Bucher
    Abstract:

    Background The red flour beetle Tribolium castaneum has developed into an insect model system second only to Drosophila. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for Tribolium research has expanded in the past years but spatio-temporally controlled Misexpression of genes has not been possible so far.